Effective treatment of esophageal adenocarcinoma using triciribine and related compounds

ABSTRACT

The inventors have determined, contrary to the prior art and experience, how to successfully use triciribine to treat esophogeal adenocarcinoma by one or a combination of (i) administering triciribine only to patients which according to a diagnostic test described below, exhibit enhanced sensitivity to the drug; (ii) use of a described dosage level that minimizes the toxicity of the drug but yet still exhibits efficacy; or (iii) use of a described dosage regimen that minimizes the toxicity of the drug.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/832,400, which was filed Aug. 21, 2015, and issued as U.S. Pat. No.9,457,040, which is a continuation of Ser. No. 13/908,821, which wasfiled Jun. 3, 2013, and issued as U.S. Pat. No. 9,150,604, which is acontinuation of U.S. application Ser. No. 13/163,123, which was filedJun. 17, 2011, and issued as U.S. Pat. No. 8,476,241, which is acontinuation of U.S. application Ser. No. 12/206,468, which was filedSep. 8, 2008, and issued as U.S. Pat. No. 8,178,502, and claims thebenefit of U.S. provisional patent application No. 60/935,940, which wasfiled Sep. 7, 2007, the disclosures of each of which is incorporatedherein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This application may claim subject matter that was developed usinggrants from the National Institute of Health and the Department ofDefense.

TECHNICAL FIELD

This application provides particular therapeutic regimens of triciribineand related compounds and compositions with reduced toxicity for thetreatment of esophogeal adenocarcinoma and other disorders associatedwith abnormal cell proliferation. A computer readable text file,entitled “1018895003US1SequenceListing.txt,” created on or about Jun.17, 2011 having a size of about 34 kb submitted in U.S. patentapplication Ser. No. 13/163,123 contains the sequence listing for thisapplication and is hereby incorporated by reference.

BACKGROUND

Cancer is an abnormal growth of cells. Cancer cells rapidly reproducedespite restriction of space, nutrients shared by other cells, orsignals sent from the body to stop reproduction. Cancer cells are oftenshaped differently from healthy cells, do not function properly, and canspread into many areas of the body. Abnormal growths of tissue, calledtumors, are clusters of cells that are capable of growing and dividinguncontrollably. Tumors can be benign (noncancerous) or malignant(cancerous). Benign tumors tend to grow slowly and do not spread.Malignant tumors can grow rapidly, invade and destroy nearby normaltissues, and spread throughout the body.

Cancers are classified according to the kind of fluid or tissue fromwhich they originate, or according to the location in the body wherethey first developed. In addition, some cancers are of mixed types.Cancers can be grouped into five broad categories, carcinomas, sarcomas,lymphomas, leukemias, and myelomas, which indicate the tissue and bloodclassifications of the cancer. Carcinomas are cancers found in bodytissue known as epithelial tissue that covers or lines surfaces oforgans, glands, or body structures. For example, a cancer of the liningof the stomach is called a carcinoma. Many carcinomas affect organs orglands that are involved with secretion, such as breasts that producemilk. Carcinomas account for approximately eighty to ninety percent ofall cancer cases. Sarcomas are malignant tumors growing from connectivetissues, such as cartilage, fat, muscle, tendons, and bones. The mostcommon sarcoma, a tumor on the bone, usually occurs in young adults.Examples of sarcoma include osteosarcoma (bone) and chondrosarcoma(cartilage). Lymphoma refers to a cancer that originates in the nodes orglands of the lymphatic system, whose job it is to produce white bloodcells and clean body fluids, or in organs such as the brain and breast.Lymphomas are classified into two categories: Hodgkin's lymphoma andnon-Hodgkin's lymphoma. Leukemia, also known as blood cancer, is acancer of the bone marrow that keeps the marrow from producing normalred and white blood cells and platelets. White blood cells are needed toresist infection. Red blood cells are needed to prevent anemia.Platelets keep the body from easily bruising and bleeding. Examples ofleukemia include acute myelogenous leukemia, chronic myelogenousleukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia.The terms myelogenous and lymphocytic indicate the type of cells thatare involved. Finally, myelomas grow in the plasma cells of bone marrow.In some cases, the myeloma cells collect in one bone and form a singletumor, called a plasmacytoma. However, in other cases, the myeloma cellscollect in many bones, forming many bone tumors. This is called multiplemyeloma.

Tumor induction and progression are often the result of accumulatedchanges in the tumor-cell genome. Such changes can include inactivationof cell growth inhibiting genes, or tumor suppressor genes, as well asactivation of cell growth promoting genes, or oncogenes. Hundreds ofactivated cellular oncogenes have been identified to date in animalmodels, however, only a small minority of these genes have proven to berelevant to human cancers (Weinberg et al 1989 Oncogenes and theMolecular Origins of Cancer Cold Spring Harbor, N.Y., Stanbridge andNowell 1990 Cell 63 867-874, Godwin et al 1992 Oncogenes andantioncogenes in gynecological malignancies. In W J Hoskins, C A Perezand R C Young (eds), Gynecological oncology: principles and practice, pp87-116, Lippincott, Philadelphia). The activation of oncogenes in humancancers can result from factors such as increased gene copy number orstructural changes. These factors can cause numerous cellular effects,for example, they can result in overexpression of a gene product.Several oncogenes involved in human cancer can be activated through geneoverexpression.

It has become apparent that the successive genetic aberrations acquiredby cancer cells result in defects in regulatory signal transductioncircuits that govern normal cell proliferation, differentiation andprogrammed cell death (Hanahan, D. and R. A. Weinberg, Cell, 2000.100(1): p. 57-700). This in turn results in fundamental defects in cellphysiology which dictate malignancy. These defects include: a) selfsufficiency in growth signals (i.e. overexpression of growth factorreceptor tyrosine kinases such as EGFR and aberrant activation ofdownstream signal transduction pathways such as Ras/Raf/Mek/Erk ½ andRas/PI3K/Akt), b) resistance to anti-growth signals (i.e. lowerexpression of TGFβ and its receptor), c) evading apoptosis (i.e. loss ofproapoptotic p53; overexpression of pro-survival Bcl-2; hyperactivationof survival pathways such as those mediated by PI3K/Akt), d) sustainedangiogenesis (i.e. high levels of secretion of VEGF) and f) tissueinvasion and metastasis (i.e. extracellular proteases and prometastaticintegrins) (Hanahan, D. and R. A. Weinberg, Cell, 2000. 100(1): p.57-700).

Receptor tyrosine kinases such as EGFR, ErbB2, VEGFR and insulin-likegrowth factor I receptor (IGF-1R) are intimately involved in thedevelopment of many human cancers including colorectal pancreatic,breast and ovarian cancers (Khaleghpour, K., et al. Carcinogenesis,2004. 25(2): p. 241-8.; Sekharam, M., et al., Cancer Res, 2003. 63(22):p. 7708-16). Binding of ligands such as EGF, VEGF and IGF-1 to theirreceptors promotes stimulation of the intrinsic tyrosine kinaseactivity, autophosphorylation of specific tyrosines in the cytoplasmicdomain of the receptors and recruitment of signaling proteins thattrigger a variety of complex signal transduction pathways (Olayioye, M.A., et al., Embo J, 2000. 19(13): p. 3159-67, Porter, A. C. and R. R.Vaillancourt, Oncogene, 1998. 17(11 Reviews): p. 1343-52). This in turnleads to the activation of many tumor survival and oncogenic pathwayssuch as the Ras/Raf/Mek/Erk ½, JAK/STAT3 and PI3K/Akt pathways. Althoughall three pathways have been implicated in colon, pancreatic, breast andovarian oncogenesis, those that are mediated by Akt have been shown tobe critical in many steps of malignant transformation including cellproliferation, anti-apoptosis/survival, invasion and metastasis andangiogenesis (Datta, S. R. et al. Genes Dev, 1999. 13(22): p. 2905-27).

Akt is a serine/threonine protein kinase (also known as PK_(B)), whichhas 3 family members Akt1, Akt2 and Akt3. Stimulation of cells withgrowth or survival factors results in recruitment to the receptors ofthe lipid kinase phosphoinositide-3-OH-kinase (PI3K) whichphosphorylates phosphoinositol-4,5-biphosphate (PIP₂) to PIP₃ whichrecruits Akt to the plasma membrane where it can be activated byphosphorylation on Thr308 and Ser473 (Akt1), Thr308 and Ser474 (Akt2)and Thr308 and Ser472 (Akt3) (Datta, S. R. et al. Genes Dev, 1999.13(22): p. 2905-27). Thus, PI3K activates Akt by phosphorylating PIP2and converting to PIP3. The phosphatase PTEN dephophorylates PIP3 toPIP2 and hence prevents the activation of Akt.

The majority of human cancers contain hyperactivated Akt (Datta, S. R.et al. Genes Dev, 1999. 13(22): p. 2905-27, Bellacosa, A., et al., Int JCancer, 1995. 64(4): p. 280-5; Sun, M., et al., Am J Pathol, 2001.159(2): p. 431-7). In particular, Akt is overexpressed and/orhyperactivated in 57%, 32%, 27% and 36% of human colorectal, pancreatic,breast and ovarian cancers, respectively (Roy, H. K., et al.Carcinogenesis, 2002. 23(1): p. 201-5, Altomare, D. A., et al., J CellBiochem, 2003. 88(1): p. 470-6., Sun, M., et al., Cancer Res, 2001.61(16): p. 5985-91., Stal, O., et al. Breast Cancer Res, 2003. 5(2): p.R37-44, Cheng, J. Q., et al., Proc Natl Acad Sci USA, 1992. 89(19): p.9267-71, Yuan, Z. Q., et al., Oncogene, 2000. 19(19): p. 2324-30).Hyperactivation of Akt is due to amplification and/or overexpression ofAkt itself as well as genetic alterations upstream of Akt includingoverexpression of receptor tyrosine kinases and/or their ligands(Khaleghpour, K., et al. Carcinogenesis, 2004. 25(2): p. 241-8.;Sekharam, M., et al., Cancer Res, 2003. 63(22): p. 7708-16, Cohen, B.D., et al., Biochem Soc Symp, 1998. 63: p. 199-210., Muller, W. J., etal. Biochem Soc Symp, 1998. 63: p. 149-57, Miller, W. E., et al. JVirol, 1995. 69(7): p. 4390-8, Slamon, D. J., et al., Science, 1987.235(4785): p. 177-82, Andrulis, I. L., et al., J Clin Oncol, 1998.16(4): p. 1340-9) and deletion of the phosphatase PTEN. Proof-of-conceptof the involvement of Akt in oncogenesis has been demonstratedpreclinically by showing that ectopic expression of Akt inducesmalignant transformation and promotes cell survival (Sun, M., et al. AmJ Pathol, 2001. 159(2): p. 431-7, Cheng, J. Q., et al., Oncogene, 1997.14(23): p. 2793-801) and that disruption of Akt pathways inhibits cellgrowth and induces apoptosis (Jetzt, A., et al. Cancer Res, 2003.63(20): p. 6697-706).

Current treatments of cancer and related diseases have limitedeffectiveness and numerous serious unintended side effects. Despitedemonstrated clinical efficacy of many anti-cancer drugs, severesystemic toxicity often halts the clinical development of promisingchemotherapeutic agents. Further, overexpression of receptor tyrosinekinases such as EGFR and their ligands such as IGF-1, Akt overexpressionand/or loss of PTEN (all of which result in hyperactivation of Akt) areassociated with poor prognosis, resistance to chemotherapy and shortenedsurvival time of cancer patients. Current research strategies emphasizethe search for effective therapeutic modes with less risk.

Triciribine

The anticancer action of triciribine (TCN, NSC-154202,3-amino-1,5-dihydro-5-methyl-1-β-ribofuranosyl-1,4,5,6,8-pentaazaacenaphthylene)and its 5′-phosphate ester, triciribine phosphate (TCN-P, NSC-280594)was initially identified in the 1970s (Townsend & Milne (1975) Ann NYAcad Sci, 255: 92-103). TCN-P was the chemical entity advanced intoclinical trials because it is more soluble than the parent drug. By theearly eighties, TCN-P had shown preclinical activity against leukemiasand carcinomas. By the early eighties, TCN-P had been identified as aninhibitor of DNA, RNA and protein synthesis, which demonstratedselectivity towards cells in the S phase of the cell cycle (Roti-Roti etal. 1978 Proc Am Assoc Cancer Res and ASCO 19:40). It had also beenproposed that unlike other nucleoside antitumor agents at the time,TCN-P is not phosphorylated beyond the level of the monophosphate and isnot incorporated into polynucleotides (Bennett et al 1978 BiochemPharmacol 27:233-241, Plagemann JNCI 1976 57: 1283-95). It was alsoestablished that in vivo, TCN-P is dephosphorylated to TCN by a plasmaenzyme and by cellular ecto-5′-nucleotidase. Inside the cells, TCN canbe rephosphorylated to TCN-P by adenosine kinase (Wotring et al 1981Proc Am Assoc Cancer Res 22: 257, Basseches et al. J Chromatogr 1982233: 227-234).

In 1982, TCN-P was entered into Phase I clinical trials by Mittelman andcolleagues in twenty patients with advanced refractory malignancies(Mittelman et al. 1983 Cancer Treat Rep 67: 159-162). TCN-P wasadministered as an intravenous (i.v.)

infusion over fifteen minutes once every three weeks at doses from 25 to350 mg/m². The patients in the trial were diagnosed with breast,head/neck, lung, pancreas, thyroid, melanoma or undetermined cancer.Only limited therapeutic responses were found and significant toxicitywas evident. Mittelman's group concluded that further clinical trialsemploying their dosing schedule were not warranted, but urged othergroups to examine the effects of TCN-P in certain specific cancer types.Also in 1983, Lu et al. (ASCO Abstracts, Clinical Pharmacology, p 34C-133) examined the clinical pharmacology of TCN in patients given 30-40mg/m² intravenously by continuous infusion for five days. Lu et al.reported that TCN contributed to liver toxicity and anemia and suggestedthat patients should be monitored for these toxicities.

Cobb et al (Cancer Treat Reports 1983 67: 173) reported the activity ofTCN-P against surgical explants of human tumors in the six day subrenalcapsule assay in mice. They examined eighty tumor types that representedbreast, lung, colon, ovarian and cervical. Cobb et al reported that TCNproduced variable response rates in the different tumors, ranging from21% (breast) to 88% (cervical).

Another Phase I was also reported by Feun et al. in 1984 (CancerResearch 44 (8) 3608-12). Feun et al administered 10, 20, 30, and/or 40mg/m² intravenously by continuous infusion for five days, every three tofour or six weeks. The patients in the trial had been diagnosed withcolon, sarcoma, melanoma, lung or “other” cancer. Feun et al. reportedthat significant toxicity was seen, including hyperglycemia,hepatotoxicity and thrombocytopenia. The authors recommended a schedulefor Phase II trials of 20 mg/m² per day for five days for six weeks andalso recommended due to the toxicity that the patients should be closelymonitored for liver and pancreatic function, and that patients withdiabetes, liver dysfunction or massive hepatic metastasis should beexcluded.

In 1986, Schilcher et al. (Cancer Research 1986 46: 3147-3151) reportedthe results of a Phase I evaluation of TCN-P using a weekly intravenousregimen. The study was conducted in twenty-four patients with advancedsolid cancers via a slow intravenous injection over five minutes on days1, 8, 15 and 22 of a 42 day cycle with a two week rest. Five dose levelsranging from 12 to 96 mg/m² were studied with 3-12 patients treated ateach level with a total of 106 doses administered. The patients in thetrial had been diagnosed with colon, rectal, bladder, adrenal, ovarian,pancreas, sarcoma, melanoma, lung or “other” cancer. Schilcher et al.concluded

“This weekly schedule produced unexpected clinical toxicity and shouldnot be pursued.”

“At this time our group is discouraged to conduct further studies withTCN-P given on weekly or intermittent schedules. A future Phase I-IIstudy using a different regimen (e.g., a single application once amonth) might be resumed if TCN-P demonstrates a pronounced in vitroactivity against therapeutic resistant primary pancreatic and hepatictumors.”

In 1986, Powis et al (Cancer Treatment Reports 70: 359-362) reported thedisposition of TCN-P in blood and plasma of patients during Phase I andII clinical trials. The Phase I trial employed a daily dose of 24-55mg/m² for 5 days, whereas the Phase II clinical trial employed a singledose of 250 mg/m². Powis et al failed to identify a correlation betweenTCN-P pharmacokinetic parameters and toxicity of TCN-P.

In the late 1980s, early 1990s, TCN-P advanced to Phase II trials formetastatic colorectal adenocarcinoma, non-small cell lung cancer,advanced squamous call carcinoma of the cervix and metastatic breastcancer. In 1987, O'Connell et al. (Cancer Treat Reports 71, No. 3,333-34) published the results of a Phase II trial in patients withmetastatic colorectal adenocarcinoma. The patients were administeredTCN-P i.v. over 15 minutes 165 or 250 mg/m² once a week in three weekintervals. O'Connell et al. concluded that the trials show a lack ofclinical usefulness of TCN-P in the treatment of patients withmetastatic colorectal adenocarcinoma. Further, in 1991, Lyss, et al.,(Proc Annu Meet Am Soc Clin Oncol, (1996) 15 A1151) reported thepreliminary results of a trial of the administration of 35 mg/m² per dayfor five days once every six weeks to patients with advanced non-smallcell lung cancer.

Feun et al. (Am J Clin Oncol 1993 16: 506-508) reported the results of aPhase II trial of TCN-P in patients with advanced squamous cellcarcinoma of the cervix. A 5 day continuous infusion of at least 35mg/m² was repeated every six weeks. Among the twenty-one evaluablepatients, only two responses were observed. Fuen et al. concluded “usingthis dose and schedule, TCN-P appears to have limited activity inmetastatic or recurrent squamous cell cancer of the cervix.”

In 1996, Hoffman et al (Cancer Chemother Pharmacol 37: 254-258) reportedthe results of a Phase I-II study of TCN-P for metastatic breast cancer.In one study, fourteen patients were treated with 20 mg/m² per day viacontinuous infusion for five days every six weeks. When the authorsfailed to see a response at this dose, the dose was escalated to atleast 35 mg/m² using the same 5 day continuous infusion schedule.Hoffman et al concluded that “TCN is ineffective at all doses tested andat doses of greater than or equal to 35 mg/m² has unacceptable toxiceffects.”

Thus, the combination of limited efficacy and unacceptable toxicityprevented the further clinical development of TCN-P and relatedcompounds.

WO 03/079748 to the Regents of the University of California disclosedcertain ZNF217 inhibitors, such as triciribine, in combination withadditional chemotherapeutic agents, such as doxorubicon.

It is an object of the present invention to provide for theadministration of triciribine and related compounds and compositionswith reduced toxicity for the treatment of tumors, cancer, and othersdisorders associated with abnormal cell proliferation.

It is another object of the present invention to provide improvedmethods to treat tumors or cancer in the subject with triciribine andrelated compounds.

SUMMARY OF THE INVENTION

The present invention provides novel therapeutic regimens oftriciribine, triciribine phosphate and related compounds to treatesophogeal adenocarcinoma in a subject while limiting systemic toxicity.The invention is based on the discovery that esophogeal adenocarcinoma,which overexpress Akt kinase are particularly sensitive to the cytotoxiceffects of TCN and related compounds. The inventors have determined,contrary to the prior art and experience, how to successfully usetriciribine to treat esophogeal adenocarcinoma by one or a combinationof (i) administering triciribine only to patients which according to adiagnostic test described below, exhibit enhanced sensitivity to thedrug; (ii) use of a described dosage level that minimizes the toxicityof the drug but yet still exhibits efficacy; or (iii) use of a describeddosage regimen that minimizes the toxicity of the drug.

In one aspect of the present invention, methods are provided to identifyesophogeal adenocarcinoma susceptible to the toxic effects of TCN, TCN-Pand/or related compounds. In one embodiment, methods are provided fortreating esophogeal adenocarcinoma in a mammal, particularly a humanthat includes (i) obtaining a biological sample from the tumor; (ii)determining whether the tumor overexpresses an Akt kinase, and (iii)treating the tumor that overexpresses Akt kinase with triciribine,triciribine phosphate or a related compound as described herein. In oneembodiment, the level of Akt kinase expression can be determined byassaying the esophogeal adenocarcinoma for the presence of aphosphorylated Akt kinase, for example, by using an antibody that candetect the phosphorylated form. In another embodiment, the level of Aktexpression can be determined by assaying a esophogeal adenocarcinomacell obtained from a subject and comparing the levels to a controltissue. In certain embodiments, the Akt can be overexpressed at least 2,2.5, 3 or 5 fold in the esophogeal adenocarcinoma sample compared to thecontrol. In certain embodiments, the overexpressed Akt kinase can be ahyperactivated and phosphorylated Akt kinase.

In another aspect of the present invention, dosing regimens are providedthat limit the toxic side effects of TCN and related compounds. In oneembodiment, such dosing regimens minimize or eliminate toxic sideeffects, including, but not limited to, hepatoxicity, thrombocytopenia,hyperglycemia, vomiting, hypocalcemia, anemia, hypoalbunemia,myelosuppression, hypertriglyceridemia, hyperamylasemia, diarrhea,stomachitis and/or fever. In another embodiment, the administration ofTCN, TCN-P or related compounds provides at least a partial, such as atleast 15, 20 or 30%, or complete response in vivo in at least 15, 20, or25% of the subjects.

In one embodiment, a method is provided to treat a subject which hasbeen diagnosed with esophogeal adenocarcinoma by administering to thesubject an effective amount of TCN, TCN-P or a related compound, forexample compounds described herein, according to a dosing schedule thatincludes administering the drug approximately one time per week forapproximately three weeks followed by a one week period wherein the drugis not administered. In another embodiment, methods are provided totreat esophogeal adenocarcinoma in a subject by administering to thesubject a dosing regimen of 10 mg/m² or less of TCN, TCN-P or a relatedcompound one time per week. In one embodiment, the compound can beadministered as a single bolus dose over a short period of time, forexample, about 5, 10 or 15 minutes. In further embodiments, dosingschedules are provided in which the compounds are administered viacontinuous infusion for at least 24, 48, 72, 96, or 120 hours. Incertain embodiments, the continuous administration can be repeated atleast once a week, once every two weeks and/or once a month. In otherembodiments, the compounds can be administered at least once every threeweeks. In further embodiments, the compounds can be administered atleast once a day for at least 2, 3, 4 or 5 days.

In further embodiments, TCN, TCN-P and related compounds as disclosedherein can be administered to patients in an amount that is effective incausing esophogeal adenocarcinoma regression. The administration of TCN,TCN-P or related compounds can provide at least a partial, such as atleast 15, 20 or 30%, or complete response in vivo in at least 15-20% ofthe subjects. In certain embodiments, at least 2, 5, 10, 15, 20, 30 or50 mg/m² of a compound disclosed herein can be administered to asubject. The administration of the compound can be conducted accordingto any of the therapeutic regimens disclosed herein. In particularembodiments, the dosing regimen can include administering less than 20mg/m² of TCN and related compounds. In one embodiment, less than 10mg/m² of TCN or related compounds can be administered once a week. Infurther embodiments, dosages of or less than 2 mg/m², 5 mg/m², 10 mg/m²,and/or 15 mg/m² of TCN or a related compound can be administered to asubject. In another embodiment, less than 10 mg/m² can be administeredto a subject via continuous infusion for at least five days. Inparticular embodiments, TCN or a related compound as disclosed hereincan be used for the treatment of esophogeal adenocarcinoma.

In one embodiment, the compounds and/or therapeutic regimens of thepresent invention can be used to prevent and/or treat esophogealadenocarcinoma. In particular embodiments, TCN or a related compound asdisclosed herein can be used for the treatment of esophogealadenocarcinoma. In further embodiments of the present invention, thecompounds disclosed herein can be used in the treatment ofangiogenesis-related diseases. In certain embodiments, methods areprovided to treat esophogeal adenocarcinoma via continuous infusion ofTCN, TCN-P or a related compound via continuous infusion for at least24, 48, 72 or 96 hours. In other embodiments, the continuous infusioncan be repeated, for example, at least once every two, three or fourweeks.

In a particular embodiment, there is provided a method for the treatmentof esophogeal adenocarcinoma, and others disorders associated with anabnormal cell proliferation in a host, the method comprisingadministering to the host an effect amount of a compound disclosedherein optionally in combination with a pharmaceutically acceptablecarrier.

In one aspect, the compounds and compositions can be administered incombination or alternation with at least one additional chemotherapeuticagent. The drugs can form part of the same composition, or be providedas a separate composition for administration at the same time or adifferent time. In one embodiment, compositions of the invention can becombined with antiangiogenic agents. In other embodiments of the presentinvention, the compounds and compositions disclosed herein can be usedin combination or alternation with the following types of drugs,including, but not limited to: antiproliferative drugs, antimitoticagents, antimetabolite drugs, alkylating agents or nitrogen mustards,drugs which target topoisomerases, drugs which target signaltransduction in tumor cells, gene therapy and antisense agents, antibodytherapeutics, steroids, steroid analogues, anti-emetic drugs and/ornonsteroidal agents.

In other embodiments, TCN, TCN-P or a related compound as disclosedherein can be used to treat esophogeal adenocarcinoma resistant to oneor more drugs, including the embodiments of esophogeal adenocarcinomaand drugs disclosed herein. In one embodiment, TCN, TCN-P or a relatedcompound as disclosed herein is administered in an effective amount forthe treatment of a patient with a drug resistant esophogealadenocarcinoma, for example, multidrug resistant esophogealadenocarcinoma, including but not limited to those resistant to taxol,rapamycin, tamoxifen, cisplatin, and/or gefitinib (iressa). In oneembodiment, the TCN, TCN-P or related compound as disclosed herein canbe administered with an additional chemotherapeutic agent that can be aP-glycoprotein inhibitor, such as verapamil, cyclosporin (such ascyclosporin A), tamoxifen, calmodulin antagonists, dexverapamil,dexniguldipine, valspodar (PSC 833), biricodar (VX-710), tariquidar(XR9576), zosuquidar (LY335979), laniquidar (R101933), and/or ONT-093.

In certain embodiments, a method is provided including administering toa host in need thereof an effective amount of a compound disclosedherein, or pharmaceutical composition comprising the compound, in aneffective amount for the treatment of the treatment of tumors, cancer,and others disorders associated with an abnormal cell proliferation in ahost.

In one embodiment, a method for the treatment of a esophogealadenocarcinoma is provided including an effective amount of a compounddisclosed herein, or a salt, isomer, prodrug or ester thereof, to anindividual in need thereof. The compound, or salt, isomer, prodrug orester thereof, is optionally provided in a pharmaceutically acceptablecomposition including the appropriate carriers, such as water, which isformulated for the desired route of administration to an individual inneed thereof. Optionally the compound is administered in combination oralternation with at least one additional therapeutic agent for thetreatment of esophogeal adenocarcinoma.

Also within the scope of the invention is the use of a compounddisclosed herein or a salt, prodrug or ester thereof in the treatment ofesophogeal adenocarcinoma, optionally in a pharmaceutically acceptablecarrier; and the use of a compound disclosed herein or a salt, prodrugor ester thereof in the manufacture of a medicament for the treatment ofcancer or tumor, optionally in a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1D demonstrate the identification of API-2 (triciribine) asa candidate of Akt inhibitor from the NCI Diversity Set. FIG. 1Aillustrates the chemical structure of API-2 (triciribine). FIG. 1Bdemonstrates that API-2 inhibits phosphorylation levels of AKT2 inAKT2-transformed NIH3T3 cells. While type AKT2-transformed NIH3T3 cellswere treated with API-2 (1 uM) for indicated times and subjected toimmunoblotting analysis with anti-phospho-Akt-T308 and -S473 antibodies(top and middle panels). The bottom panel shows expression of totalAKT2. In FIG. 1C, it is shown that API-2 inhibits three isoforms of Akt.HEK293 cells were transfected with HA-Akt1, -AKT2 and -AKT3 and treatedwith API-2 (1 uM) or wortmannin (15 uM) prior to EGF stimulation, thecells were lysed and immunoprecipitated with anti-HA antibody. Theimmunoprecipitates were subjected to in vitro kinase assay (top) andimmunoblotting analysis with anti-phospho-Akt-T308 (bottom) antibody.Middle panel shows expression of transfected Akt1, AKT2 and AKT3. FIG.1D illustrates that API-2 did not inhibit Akt in vitro. In vitro kinaseassay of constitutively active AKT2 recombinant protein in a kinasebuffer containing 1 uM API-2 (lane 3).

FIG. 2A-FIG. 2F demonstrate that API-2 does not inhibit PI3K, PDK1 andthe closely related members of AGC kinase family. FIG. 2A demonstratesan in vitro PI3K kinase assay. HEK293 cells were serum-starved andtreated with API-2 (1 uM) or Wortmannin (15 uM) for 30 minutes prior toEGF stimulation. Cells were lysed and immunoprecipitated with anti-p110αantibody. The immunoprecipitates were subjected to in vitro kinase assayusing PI-4-P as substrate. FIG. 2B illustrates the effect of API-2 on invitro PDK1 activation (top panel), closed circles show inhibition byAPI-2. Open circles show inhibition by the positive controlstaurosporine, which is a potent PDK1 inhibitor (IC50=5 nM). Bottompanels are immunoblotting analysis of HEK293 cells that were transfectedwith Myc-PDK1 and treated with wortmannin or API-2 prior to EGFstimulation. The immunoblots were detected with indicated antibodies.FIG. 2C illustrates an immunoblot analysis of phosphorylation levels ofPKCα with anti-phospho-PKCα-T638 (top) and total PKCα (bottom)antibodies following treatment with API-2 or a nonselective PKCinhibitor Ro31-8220. FIG. 2D shows an in vitro SGK kinase assay. HEK293cells were transfected with HA-SGK and treated with API-2 or wortmanninprior to EGF stimulation. In vitro kinase was performed with HA-SGKimmunoprecipitates using MBP as substrate (top). Bottom panel shows theexpression of transfected HA-SGK. FIG. 2E illustrates the results of aPKA kinase assay. Immuno-purified PKA was incubated in ADB buffer(Upstate Biotechnology Inc) containing indicated inhibitors (API-2 orPKAI) and substrate Kemptide. The kinase activity was quantified. InFIG. 2F, a western blot is shown. OVCAR3 cells were treated with API-2for indicated times. Cell lysates were immunoblotted with indicatedanti-phospho-antibodies (panels 1-4) and anti-actin antibody (bottom)

FIG. 3A, FIG. 3B-1-FIG. 3B-12, and FIG. 3C-1-FIG. 3C-7 demonstrate thatAPI-2 inhibits Akt activity and cell growth and induces apoptosis inhuman cancer cells with elevated Akt. FIG. 3A is a western blot,following treatment with API-2, phosphorylation levels of Akt weredetected with anti-phospho-Akt-T308 antibody in indicated human cancercell lines. The blots were reprobed with anti-total Akt antibody (bottompanels). In FIG. 3B-1-FIG. 3B-12, a cell proliferation assay is shown.Cell lines as indicated in the figure were treated with different dosesof API-2 for 24 h and 48 h and then analyzed with CellTiter 96 CellProliferation Assay kit (Promega). FIG. 3C-1-FIG. 3C-7 provides anapoptosis analysis. Cells were treated with API-2 and stained withannexin V and PI and analyzed by FACScan.

FIG. 4A, FIGS. 4B-1-4B-5, and FIG. 4C-FIG. 4E show that API-2 inhibitsdownstream targets of Akt and exhibits anti-tumor activity in cancercell lines with elevated Akt in mouse xenograft. In FIG. 4A, it isdemonstrated that API-2 inhibits Akt phosphorylation of tuberin, Bad,AFX and GSK-3β. Following treatment with API-2, OVAR3 cells were lysedand immunoblotted with indicated antibodies. FIGS. 4B-1-4B5 show thatAPI-2 inhibits tumor growth. Tumor cells were subcutaneously injectedinto nude mice with low level of Akt cells on left side and elevatedlevel of Akt cells on right side. When the tumors reached an averagesize of about 100-150 mm³, animals were treated with either vehicle or 1mg/kg/day API-2. Each measurement represents an average of 10 tumors.FIG. 4C illustrates a representation of the mice with OVCAR3 (right) andOVCAR5 (left) xenograft treated with API-2 or vehicle (control). FIG. 4Dshows examples of tumor size (bottom) and weight (top) at the end ofexperiment. In FIG. 4E, mmunoblot analysis of tumor lysates wasperformed with anti-phospho-Akt-S473 (top) and anti-AKT2 (bottom)antibodies in OVCAR-3-derived tumors that were treated (T3 and T4) anduntreated (T1 and T2) with API-2.

FIG. 5 shows that API-2 (triciribine) inhibits Akt kinase activity invitro. In vitro kinase assay was performed with recombinant of PDK1 andAkt in a kinase buffer containing phosphatidylinositol-3,4,5-P3 (PIP3),API-2 and histone H2B as substrate. After incubation of 30 min, thereactions were separated by SDS-PAGE and exposed in a film.

FIG. 6A-FIG. 6D provide the mRNA (SEQ ID NO: 1) and amino acid sequence(SEQ ID NO: 2) of human Akt1, restriction enzyme sites are also noted.

FIG. 7A-FIG. 7D provide the mRNA (SEQ ID NO: 3) and amino acid sequenceSEQ ID NO: 4) of human Akt2 restriction enzyme sites are also noted.

FIG. 8A-FIG. 8D provide the mRNA (SEQ ID NO. 5) and amino acid sequenceSEQ ID NO: 6) of human Akt3 restriction enzyme sites are also noted.

DETAILED DESCRIPTION

The inventors have determined, contrary to the prior art and experience,how to successfully use triciribine to treat esophogeal adenocarcinomaby one or a combination of (i) administering triciribine only topatients which according to a diagnostic test described below, exhibitenhanced sensitivity to the drug; (ii) using a described dosage levelthat minimizes the toxicity of the drug but yet still exhibits efficacy;or (iii) using a described dosage regimen that minimizes the toxicity ofthe drug.

Esophageal adenocarcinoma has demonstrated a rapid increase in incidenceover the last 10 years. This increase mirrors a dramatic rise in that ofBarrett esophagus, which is associated with esophageal adenocarcinoma inat least 95% of cases. In an attempt to understand the pathogenesis ofesophageal adenocarcinoma, attention has turned to the antiapoptotic andoncogenic pathways. Here it is demonstrated that Akt was frequentlyactivated in Barrett esophagus-related adenocarcinoma. Surprisingly, thelevels of Akt activation were associated with tumor progression. Afterinstitutional review board ethics approval, 60 archival tissue specimensof esophageal adenocarcinoma arising on a background of Barrettesophagus were selected for immunohistochemical staining withphosphorylated Akt (p-Akt) antibody. The slides were scored by 2independent observers. Approximately 80% of high-grade dysplasia andesophageal adenocarcinoma cases demonstrated strong to moderate Aktactivity. Sixty-two percent of Barrett mucosa revealed low Akt activity,the remaining cases being p-Akt negative. None of the low-gradedysplasia cases exhibited strong p-Akt staining, whereas only weak p-Aktactivity is seen in a portion of metaplastic Barrett mucosa, Akt ishighly activated in high-grade dysplasia and esophageal adenocarcinomaarising from Barrett esophagus. These findings suggest a role of p-Aktin the progression of Barrett esophagus to esophageal adenocarcinoma andprovide the rationale for using p-Akt inhibitor API-2/triciribine, whichis currently in clinical trial, in the treatment of esophagealadenocarcinoma.

Compounds

The present invention provides for the use of TCN, TCN-P and relatedcompounds for use in particular therapeutic regimens for the treatmentof esophogeal adenocarcinoma.

In one embodiment, the compounds provided herein have the followingstructures:

wherein each R2′, R3′ and R5′ are independently hydrogen, optionallysubstituted phosphate or phosphonate (including mono-, di-, ortriphosphate or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); amide, sulfonate ester includingalkyl or arylalkyl; sulfonyl, including methanesulfonyl and benzyl,wherein the phenyl group is optionally substituted with one or moresubstituents as for example as described in the definition of an arylgiven herein; optionally substituted arylsulfonyl; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; or cholesterol;or other pharmaceutically acceptable leaving group that, in vivo,provides a compound wherein R2′, R3′ or R5′ is independently H or mono-,di- or tri-phosphate;

wherein R^(x) and R^(y) are independently hydrogen, optionallysubstituted phosphate; acyl (including lower acyl); amide, alkyl(including lower alkyl); aromatic, polyoxyalkylene such aspolyethyleneglycol, optionally substituted arylsulfonyl; a lipid,including a phospholipid; an amino acid; a carbohydrate; a peptide; orcholesterol; or other pharmaceutically acceptable leaving group. In oneembodiment, the compound is administered as a 5′-phosphoether lipid or a5′-ether lipid.

R₁ and R₂ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.

In one embodiment, R2′ and R3′ are hydrogen. In another embodiment, R2′and R5′ are hydrogen. In yet another embodiment, R2′, R3′ and R5′ arehydrogen. In yet another embodiment, R2′, R3′, R5′, R1 and R2 arehydrogen.

In another embodiment, the compound has the following structure:

wherein R₃ is H, optionally substituted straight chained, branched orcyclic alkyl (including lower alkyl), alkenyl, or alkynyl, NH₂, NHR⁴,N(R⁴)₂, aryl, alkoxyalkyl, aryloxyalkyl, or substituted aryl; and

each R⁴ independently is H, acyl including lower acyl, alkyl includinglower alkyl such as but not limited to methyl, ethyl, propyl andcyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxyalkyl,hydroxyalkyl, or aryl. In a subembodiment, R₃ is a straight chainedC1-11 alkyl, iso-propyl, t-butyl, or phenyl.

In one embodiment, the compounds provided herein have the followingstructure:

In another embodiment, the compounds provided herein have the followingstructure:

In another embodiment, the compounds provided herein have the followingstructure:

wherein R₆ is H, alkyl, (including lower alkyl) alkenyl, alkynyl,alkoxyalkyl, hydroxyalkyl, arylalkyl, cycloalkyl, NH₂, NHR⁴, NR⁴R⁴, CF₃,CH₂OH, CH₂F, CH₂Cl, CH₂CF₃, C(Y³)₃, C(Y³)₂C(Y³)₃, C(═O)OH, C(═O)OR⁴,C(═O)-alkyl, C(═O)-aryl, C(═O)-alkoxyalkyl, C(═O)NH₂, C(═O)NHR⁴,C(═O)N(R⁴)₂, where each Y³ is independently H or halo; and

each R⁴ independently is H, acyl including lower acyl, alkyl includinglower alkyl such as but not limited to methyl, ethyl, propyl andcyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxyalkyl,hydroxyalkyl, or aryl.

In a subembodiment, R₆ is ethyl, CH₂CH₂OH, or CH₂-phenyl.

In another embodiment, the compounds provided herein have the followingstructure:

wherein R₇ is H, halo, alkyl (including lower alkyl), alkenyl, alkynyl,alkoxy, alkoxyalkyl, hydroxyalkyl, cycloalkyl, nitro, cyano, OH, OR⁴,NH₂, NHR⁴, NR⁴R⁴, SH, SR⁴, CF₃, CH₂OH, CH₂F, CH₂C1, CH₂CF₃, C(Y³)₃,C(Y³)₂C(Y³)₃, C(═O)OH, C(═O)OR⁴, C(═O)-alkyl, C(═O)-aryl,C(═O)-alkoxyalkyl, C(═O)NH₂, C(═O)NHR⁴, C(═O)N(R⁴)₂, or N₃, where eachY³ is independently H or halo; and

each R⁴ independently is H, acyl including lower acyl, alkyl includinglower alkyl such as but not limited to methyl, ethyl, propyl andcyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxyalkyl,hydroxyalkyl.

In a subembodiment, R₇ is methyl, ethyl, phenyl, chloro or NH₂.

In another embodiment, the compounds provided herein have the followingstructure:

In another embodiment, the compounds provided herein have the followingstructure:

It is to be understood that the compounds disclosed herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or be stereoisomeric ordiastereomeric mixtures. It is understood that the disclosure of acompound herein encompasses any racemic, optically active, polymorphic,or steroisomeric form, or mixtures thereof, which preferably possessesthe useful properties described herein, it being well known in the arthow to prepare optically active forms and how to determine activityusing the standard tests described herein, or using other similar testswhich are will known in the art. Examples of methods that can be used toobtain optical isomers of the compounds include the following:

physical separation of crystals—a technique whereby macroscopic crystalsof the individual enantiomers are manually separated. This technique canbe used if crystals of the separate enantiomers exist, i.e., thematerial is a conglomerate, and the crystals are visually distinct;

simultaneous crystallization—a technique whereby the individualenantiomers are separately crystallized from a solution of the racemate,possible only if the latter is a conglomerate in the solid state;

enzymatic resolutions—a technique whereby partial or complete separationof a racemate by virtue of differing rates of reaction for theenantiomers with an enzyme

ienzymatic asymmetric synthesis—a synthetic technique whereby at leastone step of the synthesis uses an enzymatic reaction to obtain anenantiomerically pure or enriched synthetic precursor of the desiredenantiomer;

chemical asymmetric synthesis—a synthetic technique whereby the desiredenantiomer is synthesized from an achiral precursor under conditionsthat produce assymetry (i.e., chirality) in the product, which may beachieved using chiral catalysts or chiral auxiliaries;

diastereomer separations—a technique whereby a racemic compound isreacted with an enantiomerically pure reagent (the chiral auxiliary)that converts the individual enantiomers to diastereomers. The resultingdiastereomers are then separated by chromatography or crystallization byvirtue of their now more distinct structural differences and the chiralauxiliary later removed to obtain the desired enantiomer;

first- and second-order asymmetric transformations—a technique wherebydiastereomers from the racemate equilibrate to yield a preponderance insolution of the diastereomer from the desired enantiomer or wherepreferential crystallization of the diastereomer from the desiredenantiomer perturbs the equilibrium such that eventually in principleall the material is converted to the crystalline diastereomer from thedesired enantiomer. The desired enantiomer is then released from thediastereomer;

kinetic resolutions—this technique refers to the achievement of partialor complete resolution of a racemate (or of a further resolution of apartially resolved compound) by virtue of unequal reaction rates of theenantiomers with a chiral, non-racemic reagent or catalyst under kineticconditions;

enantiospecific synthesis from non-racemic precursors—a synthetictechnique whereby the desired enantiomer is obtained from non-chiralstarting materials and where the stereochemical integrity is not or isonly minimally compromised over the course of the synthesis;

chiral liquid chromatography—a technique whereby the enantiomers of aracemate are separated in a liquid mobile phase by virtue of theirdiffering interactions with a stationary phase. The stationary phase canbe made of chiral material or the mobile phase can contain an additionalchiral material to provoke the differing interactions;

chiral gas chromatography—a technique whereby the racemate isvolatilized and enantiomers are separated by virtue of their differinginteractions in the gaseous mobile phase with a column containing afixed non-racemic chiral adsorbent phase;

extraction with chiral solvents—a technique whereby the enantiomers areseparated by virtue of preferential dissolution of one enantiomer into aparticular chiral solvent;

transport across chiral membranes—a technique whereby a racemate isplaced in contact with a thin membrane barrier. The barrier typicallyseparates two miscible fluids, one containing the racemate, and adriving force such as concentration or pressure differential causespreferential transport across the membrane barrier. Separation occurs asa result of the non-racemic chiral nature of the membrane which allowsonly one enantiomer of the racemate to pass through.

In some embodiments, triciribine, triciribine phosphate (TCN-P),triciribine 5′-phosphate (TCN-P), or the DMF adduct of triciribine(TCN-DMF) are provided. TCN can be synthesized by any technique known toone skilled in the art, for example, as described in TetrahedronLetters, vol. 49, pp. 4757-4760 (1971). TCN-P can be prepared by anytechnique known to one skilled in the art, for example, as described inU.S. Pat. No. 4,123,524. The synthesis of TCN-DMF is described, forexample, in INSERM, vol. 81, pp. 37-82 (1978). Other compounds relatedto TCN as described herein can be synthesized, for example, according tothe methods disclosed in Gudmundsson, K. S., et al., “Synthesis ofcarbocyclic analogs of 2′,3′-dideoxysangivamycin,2′,3′-dideoxytoyocamycin, and 2′,3′-dideoxytriciribine,” NucleosidesNucleotides Nucleic Acids, 20(10-11):1823-1830 (October-November 2001);Porcari, A. R., et al., “6-N-Acyltriciribine analogues:structure-activity relationship between acyl carbon chain length andactivity against HIV-1,” J. Med. Chem., 43(12):2457-2463 (Jun. 15,2000); Porcari, A. R., et al., “Acyclic sugar analogs of triciribine:lack of antiviral and antiproliferative activity correlate with lowintracellular phosphorylation,” Nucleosides Nucleotides,18(11-12):2475-2497 (November-December 1999), Porcari, A. R., et al.,“Deoxy sugar analogues of triciribine: correlation of antiviral andantiproliferative activity with intracellular phosphorylation,” J. Med.Chem., 43(12):2438-2448 (Jun. 15, 2000), Porcari, A. R., et al.,“Synthesis and antiviral activity of 2-substituted analogs oftriciribine,” Nucleosides Nucleotides Nucleic Acids, 22(12):2171-2193(December 2003), Porcari, A. R., et al., “An improved total synthesis oftriciribine: a tricyclic nucleoside with antineoplastic and antiviralproperties,” Nucleosides Nucleotides Nucleic Acids, 23(1-2):31-39(2004), Schweinsberg, P. D., et al. “Identification of the metabolitesof an antitumor tricyclic nucleoside (NSC-154020),” Biochem. Pharmacol.,30(18):2521-2526 (Sep. 15, 1981), Smith, K. L., et al., “Synthesis ofnew 2′-beta-C-methyl related triciribine analogues as anti-HCV agents,”Bioorg. Med. Chem. Lett., 14(13):3517-3520 (Jul. 5, 2004), Townsend, L.B., et al., “The synthesis and biological activity of certainpentaazaacenaphthylenes, hexaazaacenaphthylenes and their correspondingnucleosides,” Nucleic Acids Symp. Ser., 1986(17):41-44 (1986), and/orWotring, L. L., et al., “Mechanism of activation of triciribinephosphate (TCN-P) as a prodrug form of TCN,” Cancer Treat Rep.,70(4):491-7 (April 1986).

Pharmaceutically Acceptable Salts and Prodrugs

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compound as apharmaceutically acceptable salt may be appropriate. Pharmaceuticallyacceptable salts include those derived from pharmaceutically acceptableinorganic or organic bases and acids. Suitable salts include thosederived from alkali metals such as potassium and sodium, alkaline earthmetals such as calcium and magnesium, among numerous other acids wellknown in the pharmaceutical art. In particular, examples ofpharmaceutically acceptable salts are organic acid addition salts formedwith acids, which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including, sulfate,nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

Any of the nucleotides described herein can be administered as anucleotide prodrug to increase the activity, bioavailability, stabilityor otherwise alter the properties of the nucleoside. A number ofnucleotide prodrug ligands are known. In general, alkylation, acylationor other lipophilic modification of the mono, di or triphosphate of thenucleoside will increase the stability of the nucleotide. Examples ofsubstituent groups that can replace one or more hydrogens on thephosphate moiety are alkyl, aryl, steroids, carbohydrates, includingsugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jonesand N. Bischofberger, Antiviral Research, 27 (1995) 1-17. Any of thesecan be used in combination with the disclosed nucleosides to achieve adesired effect.

In one embodiment, the triciribine or a related compound is provided as5′-hydroxyl lipophilic prodrug. Nonlimiting examples of U.S. patentsthat disclose suitable lipophilic substituents that can be covalentlyincorporated into the nucleoside, preferably at the 5′-OH position ofthe nucleoside or lipophilic preparations, include U.S. Pat. No.5,149,794 (Sep. 22, 1992, Yatvin, et al.); U.S. Pat. No. 5,194,654 (Mar.16, 1993, Hostetler, et al.); U.S. Pat. No. 5,223,263 (Jun. 29, 1993,Hostetler, et al.); U.S. Pat. No. 5,256,641 (Oct. 26, 1993, Yatvin, etal.); U.S. Pat. No. 5,411,947 (May 2, 1995, Hostetler, et al.); U.S.Pat. No. 5,463,092 (Oct. 31, 1995, Hostetler, et al.); U.S. Pat. No.5,543,389 (Aug. 6, 1996, Yatvin, et al.); U.S. Pat. No. 5,543,390 (Aug.6, 1996, Yatvin, et al.); U.S. Pat. No. 5,543,391 (Aug. 6, 1996, Yatvin,et al.); and U.S. Pat. No. 5,554,728 (Sep. 10, 1996, Basava, et al.),all of which are incorporated herein by reference.

Foreign patent applications that disclose lipophilic substituents thatcan be attached to the triciribine or a related compound s of thepresent invention, or lipophilic preparations, include WO 89/02733, WO90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO/15132,EP 0 350 287, EP 93917054.4, and WO 91/19721.

Additional nonlimiting examples of derivatives of triciribine or arelated compounds are those that contain substituents as described inthe following publications. These derivatized triciribine or a relatedcompound s can be used for the indications described in the text orotherwise as antiviral agents, including as anti-HIV or anti-HBV agents.Ho, D. H. W. (1973) Distribution of Kinase and deaminase of1β-D-arabinofuranosylcytosine in tissues of man and mouse. Cancer Res.33, 2816-2820; Holy, A. (1993) Isopolar phosphorous-modified nucleotideanalogues. In: De Clercq (ed.), Advances in Antiviral Drug Design, Vol.I, JAI Press, pp. 179-231; Hong, C. I., Nechaev, A., and West, C. R.(1979a) Synthesis and antitumor activity of1β-3-arabinofuranosylcytosine conjugates of cortisol and cortisone.Biochem. Biophys. Rs. Commun. 88, 1223-1229; Hong, C. I., Nechaev, A.,Kirisits, A. J. Buchheit, D. J. and West, C. R. (1980) Nucleosideconjugates as potential antitumor agents. 3. 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(1991) Synthesis and anti-HIVactivity of some haloalky phosphoramidate derivatives of3′-azido-3′deoxythylmidine (AZT); potent activity of the trichloroethylmethoxyalaninyl compound. Antiviral Res. 15, 255-263; McGuigan, C.,Pathirana, R. N., Mahmood, N., Devine, K. G. and Hay, A. J. (1992) Arylphosphate derivatives of AZT retain activity against HIV1 in cell lineswhich are resistant to the action of AZT. Antiviral Res. 17, 311-321;McGuigan, C., Pathirana, R. N., Choi, S. M., Kinchington, D. andO'Connor, T. J. (1993a) Phosphoramidate derivatives of AZT as inhibitorsof HIV; studies on the caroxyl terminus. Antiviral Chem. Chemother. 4,97-101; McGuigan, C., Pathirana, R. N., Balzarini, J. and De Clercq, E.(1993b) Intracellular delivery of bioactive AZT nucleotides by arylphosphate derivatives of AZT. J. Med. Chem. 36, 1048-1052.

Alkyl hydrogen phophonate derivatives of the anti-HIV agent AZT may beless toxic than the parent nucleoside analogue. Antiviral Chem.Chemother. 5, 271-277; Meyer, R. B., Jr., Shuman, D. A. and Robins, R.K. (1973) Synthesis of purine nucleoside 3′,5′-cyclic phosphoramidates.Tetrahedron Lett. 269-272; Nagyvary, J. Gohil, R. N., Kirchner, C. R.and Stevens, J. D. (1973) Studies on neutral esters of cyclic AMP,Biochem. Biophys. Res. Commun. 55, 1072-1077; Namane, A. Goyette, C.,Fillion, M. P., Fillion, G. and Huynh-Dinh, T. (1992) Improved braindelivery of AZT using a glycosyl phosphotriester prodrug. J. Med. Chem.35, 3939-3044; Nargeot, J. Nerbonne, J. M. Engels, J. and Leser, H. A.(1983) Natl. Acad. Sci. U.S.A. 80, 2395-2399; Nelson, K. A., Bentrude,W. G., Stser, W. N. and Hutchinson, J. P. (1987) The question ofchair-twist equilibria for the phosphate rings of nucleoside cyclic3′,5′-monophosphates. ¹HNMR and x-ray crystallographic study of thediasteromers of thymidine phenyl cyclic 3′,5′-monophosphate. J. Am.Chem. Soc. 109, 4058-4064; Nerbonne, J. M., Richard, S., Nargeot, J. andLester, H. A. (1984) New photoactivatable cyclic nucleotides produceintracellular jumps in cyclic AMP and cyclic GMP concentrations. Nature301, 74-76; Neumann, J. M., Herve, M., Debouzy, J. C., Guerra, F. I.,Gouyette, C., Dupraz, B. and Huynh-Dinh, T. (1989) Synthesis andtransmembrane transport studies by NMR of a glucosyl phospholipid ofthymidine. J. Am. Chem. Soc. 111, 4270-4277; Ohno, R., Tatsumi, N.,Hirano, M., Imai, K. Mizoguchi, H., Nakamura, T., Kosaka, M., Takatuski,K., Yamaya, T., Toyama, K., Yoshida, T., Masaoka, T., Hashimoto, S.,Ohshima, T., Kimura, I., Yamada, K. and Kimura, J. (1991) Treatment ofmyelodyspastic syndromes with orally administered1-β-D-rabinofuranosylcytosine-5′-stearylphosphate. Oncology 48, 451-455.Palomino, E., Kessle, D. and Horwitz, J. P. (1989) A dihydropyridinecarrier system for sustained delivery of 2′,3′dideoxynucleosides to thebrain. J. Med. Chem. 32, 622-625; Perkins, R. M., Barney, S., Wittrock,R., Clark, P. H., Levin, R. Lambert, D. M., Petteway, S. R.,Serafinowska, H. T., Bailey, S. M., Jackson, S., Harnden, M. R., Ashton,R., Sutton, D., Harvey, J. J. and Brown, A. G. (1993) Activity ofBRL47923 and its oral prodrug, SB203657A against a rauscher murineleukemia virus infection in mice. Antiviral Res. 20 (Suppl. I). 84;Piantadosi, C., Marasco, C. J., Jr., Morris-Natschke, S. L., Meyer, K.L., Gumus, F., Surles, J. R., Ishaq, K. S., Kucera, L. S. Iyer, N.,Wallen, C. A., Piantadosi, S. and Modest, E. J. (1991) Synthesis andevaluation of novel ether lipid nucleoside conjugates for anti-HIV-1activity. J. Med. Chem. 34, 1408-1414; Pompon, A., Lefebvre, I., Imbach,J. L., Kahn, S. and Farquhar, D. (1994) Decomposition pathways of themono- and bis(pivaloyloxymethyl) esters ofazidothymidine-5′-monophosphate in cell extract and in tissue culturemedium; an application of the on-line ISRP-cleaning' HPLC technique.Antiviral Chem. Chemother. 5, 91-98; Postemark, T. (1974) Cyclic AMP andcyclic GMP. Anu. Rev. Pharmacol. 14, 23-33; Prisbe, E. J., Martin, J. C.M., McGee, D. P. C., Barker, M. F., Smee, D. F. Duke, A. E., Matthews,T. R. and Verheyden, J. P. J. (1986) Synthesis and antiherpes virusactivity of phosphate and phosphonate derivatives of9-[(1,3-dihydroxy-2-propoxy)methyl] guanine. J. Med. Chem. 29, 671-675;Pucch, F., Gosselin, G., Lefebvre, I., Pompon, A., Aubertin, A. M. Dim,A. and Imbach, J. L. (1993) Intracellular delivery of nucleosidemonophosphate through a reductase-mediated activation process. AntiviralRes. 22, 155-174; Pugaeva, V. P., Kochkeva, S. I., Mashbits, F. D. andEizengart, R. S. (1969). Toxicological assessment and health standardratings for ethylene sulfide in the industrial atmosphere. Gig. Trf.Prof. Zabol. 13, 47-48 (Chem. Abstr. 72, 212); Robins, R. K. (1984) Thepotential of nucleotide analogs as inhibitors of retroviruses andtumors. Pharm. Res. 11-18; Rosowsky, A., Kim, S. H., Ross and J. Wick,M. M. (1982) Lipophilic 5′-(alkylphosphate) esters of1-β-D-arabinofuranosylcytosine and its N⁴-acyl and 2.2′-anhydro-3′0-acylderivatives as potential prodrugs. J. Med. Chem. 25, 171-178; Ross, W.(1961) Increased sensitivity of the walker turnout towards aromaticnitrogen mustards carrying basic side chains following glucosepretreatment. Biochem. Pharm. 8, 235-240; Ryu, E. K., Ross, R. J.,Matsushita, T., MacCoss, M., Hong, C. I. and West, C. R. (1982).Phospholipid-nucleoside conjugates 3. Synthesis and preliminarybiological evaluation of 1-β-D-arabinofuranosylcytosine5′diphosphate[-], 2-diacylglycerols. J. Med. Chem. 25, 1322-1329;Saffhill, R. and Hume, W. J. (1986) The degradation of5-iododeoxyurindine and 5-bromoeoxyuridine by serum from differentsources and its consequences for the use of these compounds forincorporation into DNA. Chem. Biol. Interact. 57, 347-355; Saneyoshi,M., Morozumi, M., Kodama, K., Machida, J., Kuninaka, A. and Yoshino, H.(1980) Synthetic nucleosides and nucleotides XVI. Synthesis andbiological evaluations of a series of 1-β-D-arabinofuranosylcytosine5′-alkyl or arylphosphates. Chem. Pharm. Bull. 28, 2915-2923; Sastry, J.K., Nehete, P. N., Khan, S., Nowak, B. J., Plunkett, W., Arlinghaus, R.B. and Farquhar, D. (1992) Membrane-permeable dideoxyuridine5′-monophosphate analogue inhibits human immunodeficiency virusinfection. Mol. Pharmacol. 41, 441-445; Shaw, J. P., Jones, R. J.Arimilli, M. N., Louie, M. S., Lee, W. A. and Cundy, K. C. (1994) Oralbioavailability of PMEA from PMEAprodrugs in male Sprague-Dawley rats.9th Annual AAPS Meeting. San Diego, Calif. (Abstract). Shuto, S., Ueda,S., Imamura, S., Fukukuawa, K. Matsuda, A. and Ueda, T. (1987) A facileone-step synthesis of 5′-phosphatidylnucleosides by an enzymatictwo-phase reaction. Tetrahedron Lett. 28, 199-202; Shuto, S., Itch, H.,Ueda, S., Imamura, S., Kukukawa, K., Tsujino, M. Matsuda, A. and Ueda,T. (1988) A facile enzymatic synthesis of5′-(3-sn-phosphatidyl)nucleosides and their antileukemic activities.Chem. Pharm. Bull. 36, 209-217. One preferred phosphate prodrug group isthe S-acyl-2-thioethyl group, also referred to as “SATE.”

Additional examples of prodrugs that can be used are those described inthe following patents and patent applications: U.S. Pat. Nos. 5,614,548,5,512,671, 5,770,584, 5,962,437, 5,223,263, 5,817,638, 6,252,060,6,448,392, 5,411,947, 5,744,592, 5,484,809, 5,827,831, 5,696,277,6,022,029, 5,780,617, 5,194,654, 5,463,092, 5,744,461, 4,444,766,4,562,179, 4,599,205, 4,493,832, 4,221,732, 5,116,992, 6,429,227,5,149,794, 5,703,063, 5,888,990, 4,810,697, 5,512,671, 6,030,960,2004/0259845, U.S. Pat. No. 6,670,341, 2004/0161398, 2002/082242, U.S.Pat. No. 5,512,671, 2002/0082242, and or PCT Publication Nos WO90/11079, WO 96/39197, and/or WO 93/08807.

Definitions

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth, i.e., proliferative disorders. Examples ofsuch proliferative disorders include cancers such as carcinoma,lymphoma, blastoma, sarcoma, and leukemia, as well as other cancersdisclosed herein. More particular examples of such cancers includebreast cancer, prostate cancer, colon cancer, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, cervical cancer, ovarian cancer, livercancer, e.g., hepatic carcinoma, bladder cancer, colorectal cancer,endometrial carcinoma, kidney cancer, and thyroid cancer.

Other non-limiting examples of cancers are basal cell carcinoma, biliarytract cancer; bone cancer; brain and CNS cancer; choriocarcinoma;connective tissue cancer; esophageal cancer; eye cancer; cancer of thehead and neck; gastric cancer; intra-epithelial neoplasm; larynx cancer;lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); pancreatic cancer; retinoblastoma; rhabdomyosarcoma;rectal cancer; cancer of the respiratory system; sarcoma; skin cancer;stomach cancer; testicular cancer; uterine cancer; cancer of the urinarysystem, as well as other carcinomas and sarcomas.

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. For example, a particular cancer may becharacterized by a solid mass tumor. The solid tumor mass, if present,may be a primary tumor mass. A primary tumor mass refers to a growth ofcancer cells in a tissue resulting from the transformation of a normalcell of that tissue. In most cases, the primary tumor mass is identifiedby the presence of a cyst, which can be found through visual orpalpation methods, or by irregularity in shape, texture or weight of thetissue. However, some primary tumors are not palpable and can bedetected only through medical imaging techniques such as X-rays (e.g.,mammography), or by needle aspirations. The use of these lattertechniques is more common in early detection. Molecular and phenotypicanalysis of cancer cells within a tissue will usually confirm if thecancer is endogenous to the tissue or if the lesion is due to metastasisfrom another site.

The term alkyl, as used herein, unless otherwise specified, includes asaturated straight, branched, or cyclic, primary, secondary, or tertiaryhydrocarbon of for example C₁ to C₂₄, and specifically includes methyl,trifluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl,t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl,cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. The alkyl is optionally substituted, e.g., with oneor more substituents such as halo (F, Cl, Br or I), (e.g. CF₃,2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃ or CF₂CF₃), hydroxyl (e.g. CH₂OH), amino(e.g. CH₂NH₂, CH₂NHCH₃ or CH₂N(CH₃)₂), alkylamino, arylamino, alkoxy,aryloxy, nitro, azido (e.g. CH₂N₃), cyano (e.g. CH₂CN), sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

The term lower alkyl, as used herein, and unless otherwise specified,refers to a C₁ to C₄ saturated straight, branched, or if appropriate, acyclic (for example, cyclopropyl) alkyl group, including bothsubstituted and unsubstituted forms.

The term alkylamino or arylamino includes an amino group that has one ortwo alkyl or aryl substituents, respectively.

The term amino acid includes naturally occurring and synthetic α, β, γor δ amino acids, and includes but is not limited to, amino acids foundin proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In a preferred embodiment, the amino acid is inthe L-configuration. Alternatively, the amino acid can be a derivativeof alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl,tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl,tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl,argininyl, histidinyl, β-alanyl, β-leucinyl, β-isoleuccinyl, β-prolinyl,β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-serinyl,β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl,β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl. Whenthe term amino acid is used, it is considered to be a specific andindependent disclosure of each of the esters of a natural or syntheticamino acid, including but not limited to α, β, γ or δ glycine, alanine,valine, leucine, isoleucine, methionine, phenylalanine, tryptophan,proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartate, glutamate, lysine, arginine and histidine in the D andL-configurations.

The term “protected” as used herein and unless otherwise definedincludes a group that is added to an oxygen, nitrogen, sulfur orphosphorus atom to prevent its further reaction or for other purposes. Awide variety of oxygen and nitrogen protecting groups are known to thoseskilled in the art of organic synthesis (see Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3^(rd) Ed., John Wiley & Sons, Inc., NewYork, N.Y., 1999).

The term aryl, as used herein, and unless otherwise specified, includesphenyl, biphenyl, or naphthyl, and preferably phenyl. The aryl group isoptionally substituted with one or more moieties such as halo, hydroxyl,amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, phosphonic acid, phosphate, or phosphonate, eitherunprotected, or protected as necessary, as known to those skilled in theart, for example, as taught in Greene, et al., Protective Groups inOrganic Synthesis, John Wiley and Sons, 3^(rd) Ed., 1999.

The term alkaryl or alkylaryl includes an alkyl group with an arylsubstituent. The term aralkyl or arylalkyl includes an aryl group withan alkyl substituent.

The term halo, as used herein, includes chloro, bromo, iodo, and fluoro.

The term acyl includes a carboxylic acid ester in which the non-carbonylmoiety of the ester group is selected from straight, branched, or cyclicalkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkylincluding benzyl, aryloxyalkyl such as phenoxymethyl, aryl includingphenyl optionally substituted with halogen, C₁ to C₄ alkyl or C₁ to C₄alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl includingmethanesulfonyl, the mono, di or triphosphate ester, trityl ormonomethoxytrityl, substituted benzyl, trialkylsilyl (e.g.dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in the estersoptimally comprise a phenyl group. The term “lower acyl” refers to anacyl group in which the non-carbonyl moiety is lower alkyl.

As used herein, the term “substantially free of” or “substantially inthe absence of” with respect to enantiomeric purity, refers to acomposition that includes at least 85% or 90% by weight, preferably 95%to 98% by weight, and even more preferably 99% to 100% by weight, of thedesignated enantiomer. In a preferred embodiment, in the methods andcompounds of this invention, the compounds are substantially free ofother enantiomers.

Similarly, the term “isolated” refers to a compound composition thatincludes at least 85% or 90% by weight, preferably 95% to 98% by weight,and even more preferably 99% to 100% by weight, of the compound, theremainder comprising other chemical species or enantiomers.

The term “independently” is used herein to indicate that the variable,which is independently applied, varies independently from application toapplication. Thus, in a compound such as R″XYR″, wherein R″ is“independently carbon or nitrogen,” both R″ can be carbon, both R″ canbe nitrogen, or one R″ can be carbon and the other R″ nitrogen.

The term “pharmaceutically acceptable salt or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform (such as an ester, phosphate ester, salt of an ester or a relatedgroup) of a compound, which, upon administration to a patient, providesthe compound. Pharmaceutically acceptable salts include those derivedfrom pharmaceutically acceptable inorganic or organic bases and acids.Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart. Pharmaceutically acceptable prodrugs refer to a compound that ismetabolized, for example hydrolyzed or oxidized, in the host to form thecompound of the present invention. Typical examples of prodrugs includecompounds that have biologically labile protecting groups on afunctional moiety of the active compound. Prodrugs include compoundsthat can be oxidized, reduced, aminated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated, phosphorylated, dephosphorylated to produce theactive compound.

The term “pharmaceutically acceptable esters” as used herein, unlessotherwise specified, includes those esters of one or more compounds,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of hosts without undue toxicity, irritation,allergic response and the like, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

The term “subject” as used herein refers to an animal, preferably amammal, most preferably a human. Mammals can include non-human mammals,including, but not limited to, pigs, sheep, goats, cows (bovine), deer,mules, horses, monkeys and other non-human primates, dogs, cats, rats,mice, rabbits or any other known or disclosed herein.

II. In Vivo Efficacy/Dosing Regimens

In another aspect of the present invention, dosing regimens are providedthat limit the toxic side effects of TCN and related compounds. In oneembodiment, such dosing regimens minimize the following toxic sideeffects, including, but not limited to, hepatoxicity, thrombocytopenia,hyperglycemia, vomiting, hypocalcemia, anemia, hypoalbunemia,myelosuppression, hypertriglyceridemia, hyperamylasemia, diarrhea,stomachitis and/or fever.

In another embodiment, the administration of TCN, TCN-P or relatedcompounds provides at least a partial or complete response in vivo in atleast 15-20% of the subjects. In particular embodiments, a partialreponse can be at least 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75,80 or 85% regression of the tumor. In other embodiments, this responsecan be evident in at least 15, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65,70, 75, 80, 85 or 90% of the subjects treated with the therapy. Infurther embodiments, such response rates can be obtained by anytherapeutic regimen disclosed herein.

In other embodiments, methods are provided to treat a subject that hasbeen diagnosed with esophogeal adenocarcinoma by administering to thesubject an effective amount of TCN, TCN-P or a related compoundaccording to a dosing schedule that includes administering the drug onetime per week for three weeks followed by a one week period wherein thedrug is not administered (i.e. via a 28 day cycle). In otherembodiments, such 28 day cycles can be repeated at least 2, 3, 4, or 5times or until regression of the tumor is evident.

In further embodiments, a 42 day cycle is provided in which thecompounds disclosed herein can be administered once a week for fourweeks followed by a two week period in which the drug is notadministered. In other embodiments, such 42 day cycles can be repeatedat least 2, 3, 4, or 5 times or until regression of the tumor isevident. In a particular embodiment, less than 12, less than 11 or lessthan 10 mg/m² of TCN, TCN-P or a related compound can be administeredaccording to a 42 day cycle. In other particular embodiments, 2, 3, 4,5, 6, 7, 8, 9, 10 or 11 mg/m² of TCN, TCN-P or a related compound can beadministered according to a 42 day cycle.

In another embodiment, methods are provided to treat esophogealadenocarcinoma in a subject by administering to the subject a dosingregimen of 10 mg/m² or less of TCN, TCN-P or a related compound one timeper week. In particular embodiments, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg/m² of TCN, TCN-P ora related compound as dislosed herein can be administered one time perweek

In embodiments of the present invention, the compound disclosed hereincan be administered as a single bolus dose over a short period of time,for example, about 5, 10, 15, 20, 30 or 60 minutes. In furtherembodiments, dosing schedules are provided in which the compounds areadministered via continuous infusion for at least 24, 48, 72, 96, or 120hours. In certain embodiments, the administration of the drug viacontinuous or bolus inhections can be repeated at a certain frequency atleast: once a week, once every two weeks, once every three weeks, once amonth, once every five weeks, once every six weeks, once every eightweeks, once every ten weeks and/or once every twelve weeks. The type andfrequency of administrations can be combined ion any manner disclosedherein to create a dosing cycle. The drugs can be repeatedlyadministered via a certain dosing cycles, for example as a bolusinjection once every two weeks for three months. The dosing cycles canbe administered for at least: one, two three, four five, six, seven,eight, nine, ten, eleven, twelve, eighteen or twenty four months.Alternatively, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20dosing cycles can be administered to a patient. The drug can beadministered according to any combination disclosed herein, for example,the drug can be administered once a week every three weeks for 3 cycles.

In further embodiments, the compounds can be administered at least oncea day for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Suchadministration can be followed by corresponding periods in which thedrug is not administered.

The TCN, TCN-P and related compounds as disclosed herein can beadministered to patients in an amount that is effective in causing tumorregression. The administration of TCN, TCN-P or related compounds canprovide at least a partial, such as at least 15, 20 or 30%, or completeresponse in vivo in at least 15-20% of the subjects. In certainembodiments, at least 2, 5, 10, 15, 20, 30 or 50 mg/m² of a compounddisclosed herein can be administered to a subject. In certainembodiments, at least about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 165, 175, 200, 250,300, or 350 mg/m² of TCN, TCN-P or a related compound disclosed hereincan be administered to a subject.

The administration of the compound can be conducted according to any ofthe therapeutic regimens disclosed herein. In particular embodiments,the dosing regimen includes administering less than 20 mg/m² of TCN andrelated compounds. In one embodiment, less than 20 mg/m² of TCN orrelated compounds can be administered once a week. In furtherembodiments, 2 mg/m², 5 mg/m², 10 mg/m², and/or 15 mg/m² of TCN or arelated compound can be administered to a subject. In anotherembodiment, less than 10 mg/m² can be administered to a subject viacontinuous infusion for at least five days. The present inventionprovides for any combination of dosing type, frequency, number of cyclesand dosage amount disclosed herein.

III. Screening of Patient Populations

In another aspect of the present invention, methods are provided toidentify esophogeal adenocarcinoma susceptible to the toxic effects oftriciribine (TCN) and related compounds. In one embodiment, methods areprovided to esophogeal adenocarcinoma in a mammal by (i) obtaining abiological sample from the tumor; (ii) determining whether the cancer ortumor overexpresses Akt kinase or hyperactivated and phosphorylated Aktkinase, and (iii) treating the cancer or tumor with triciribine or arelated compound as described herein. In one embodiment, the biologicalsample can be a biopsy. In other embodiments, the biological sample canbe fluid, cells and/or aspirates obtained from the tumor or cancer.

The biological sample can be obtained according to any technique knownto one skilled in the art. In one embodiment, a biopsy can be conductedto obtain the biological sample. A biopsy is a procedure performed toremove tissue or cells from the body for examination. Some biopsies canbe performed in a physician's office, while others need to be done in ahospital setting. In addition, some biopsies require use of ananesthetic to numb the area, while others do not require any sedation.In certain embodiments, an endoscopic biopsy can be performed. This typeof biopsy is performed through a fiberoptic endoscope (a long, thin tubethat has a close-focusing telescope on the end for viewing) through anatural body orifice (i.e., mouth) or a small incision (i.e.,arthroscopy). The endoscope is used to view the organ in question forabnormal or suspicious areas, in order to obtain a small amount oftissue for study. Endoscopic procedures are named for the organ or bodyarea to be visualized and/or treated. The physician can insert theendoscope into the gastrointestinal tract (alimentary tract endoscopy),bladder (cystoscopy), abdominal cavity (laparoscopy), joint cavity(arthroscopy), mid-portion of the chest (mediastinoscopy), or tracheaand bronchial system (laryngoscopy and bronchoscopy).

In another embodiment, a bone marrow biopsy can be performed. This typeof biopsy can be performed either from the sternum (breastbone) or theiliac crest hipbone (the bone area on either side of the pelvis on thelower back area). The skin is cleansed and a local anesthetic is givento numb the area. A long, rigid needle is inserted into the marrow, andcells are aspirated for study; this step is occasionally uncomfortable.A core biopsy (removing a small bone ‘chip’ from the marrow) may followthe aspiration.

In a further embodiment, an excisional or incisional biopsy can beperformed on the mammal. This type of biopsy is often used when a wideror deeper portion of the skin is needed. Using a scalpel (surgicalknife), a full thickness of skin is removed for further examination, andthe wound is sutured (sewed shut with surgical thread). When the entiretumor is removed, it is referred to as an excisional biopsy technique.If only a portion of the tumor is removed, it is referred to as anincisional biopsy technique. Excisional biopsy is often the methodusually preferred, for example, when melanoma (a type of skin cancer) issuspected.

In still further embodiments, a fine needle aspiration (FNA) biopsy canbe used. This type of biopsy involves using a thin needle to remove verysmall pieces from a tumor. Local anesthetic is sometimes used to numbthe area, but the test rarely causes much discomfort and leaves no scar.FNA is not, for example, used for diagnosis of a suspicious mole, butmay be used, for example, to biopsy large lymph nodes near a melanoma tosee if the melanoma has metastasized (spread). A computed tomographyscan (CT or CAT scan) can be used to guide a needle into a tumor in aninternal organ such as the lung or liver.

In other embodiments, punch shave and/or skin biopsies can be conducted.Punch biopsies involve taking a deeper sample of skin with a biopsyinstrument that removes a short cylinder, or “apple core,” of tissue.After a local anesthetic is administered, the instrument is rotated onthe surface of the skin until it cuts through all the layers, includingthe dermis, epidermis, and the most superficial parts of the subcutis(fat). A shave biopsy involves removing the top layers of skin byshaving it off. Shave biopsies are also performed with a localanesthetic. Skin biopsies involve removing a sample of skin forexamination under the microscope to determine if, for example, melanomais present. The biopsy is performed under local anesthesia.

In particular embodiment, methods are provided to determine whether thetumor overexpresses an Akt kinase. Akt kinase overexpression can referto the phosphorylation state of the kinase. Hyperphosphorylation of Aktcan be detected according to the methods described herein. In oneembodiment, a tumor biopsy can be compared to a control tissue. Thecontrol tissue can be a normal tissue from the mammal in which thebiopsy was obtained or a normal tissue from a healthy mammal. Akt kinaseoverexpression or hyperphosphorylation can be determined if the tumorbiopsy contains greater amounts of Akt kinase and/or Akt kinasephosphorylation than the control tissue, such as, for example, at leastapproximately 1.5, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5,4.75, 5, 5.5, 6, 7, 8, 9, or10-fold greater amounts of Akt kinase thancontained in the control tissue.

In one embodiment, the present invention provides a method to detectaberrant Akt kinase expression in a subject or in a biological samplefrom the subject by contacting cells, cell extracts, serum or othersample from the subjects or said biological sample with animmunointeractive molecule specific for an Akt kinase or antigenicportion thereof and screening for the level of immunointeractivemolecule-Akt kinase complex formation, wherein an elevated presence ofthe complex relative to a normal cell is indicative of an aberrant cellthat expresses or overexpresses Akt. In one example, cells or cellextracts can be screened immunologically for the presence of elevatedlevels of Akt kinase.

In an alternative embodiment, the aberrant expression of Akt in a cellis detected at the genetic level by screening for the level ofexpression of a gene encoding an Akt kinase wherein an elevated level ofa transcriptional expression product (i.e. mRNA) compared to a normalcell is indicative of an aberrant cell. In certain embodiments,real-time PCR as well as other PCR procedures can be used to determinetranscriptional activity. In one embodiment, mRNA can be obtained fromcells of a subject or from a biological sample from a subject and cDNAoptionally generated. The mRNA or cDNA can then be contacted with agenetic probe capable of hybridizing to and/or amplifying all or part ofa nucleotide sequence encoding Akt kinase or its complementarynucleotide sequence and then the level of the mRNA or cDNA can bedetected wherein the presence of elevated levels of the mRNA or cDNAcompared to normal controls can be assessed.

Yet another embodiment of the present invention contemplates the use ofan antibody, monoclonal or polyclonal, to Akt kinase in a quantitativeor semi-quantitative diagnostic kit to determine relative levels of Aktkinase in suspected cancer cells from a patient, which can include allthe reagents necessary to perform the assay. In one embodiment, a kitutilizing reagents and materials necessary to perform an ELISA assay isprovided. Reagents can include, for example, washing buffer, antibodydilution buffer, blocking buffer, cell staining solution, developingsolution, stop solution, anti-phospho-protein specific antibodies,anti-Pan protein specific antibodies, secondary antibodies, anddistilled water. The kit can also include instructions for use and canoptionally be automated or semi-automated or in a form which iscompatible with automated machine or software. In one embodiment, aphosphor-ser-473 Akt antibody that detects the activated form of AKT(Akt phosphorylated at serine 474) can be utilized as the antibody in adiagnostic kit. See, for example, Yuan et al. (2000) “FrequentActivation of AKT2 and induction of apoptosis by inhibition ofphosphinositide-3-OH kinase/Akt pathway in human ovarian cancer,”Oncogene 19:2324-2330.

Akt Kinases

Akt, also named PKB³, represents a subfamily of the serine/threoninekinase. Three members, AKT1, AKT2, and AKT3, have been identified inthis subfamily. Akt is activated by extracellular stimuli in aPI3K-dependent manner (Datta, S. R., et al. Genes Dev. 13: 2905-2927,1999). Full activation of Akt requires phosphorylation of Thr³⁰⁸ in theactivation loop and Ser⁴⁷³ in the C-terminal activation domain. Akt isnegatively regulated by PTEN tumor suppressor. Mutations in PTEN havebeen identified in various tumors, which lead to activation of Aktpathway (Datta, S. R., et al. Genes Dev. 13: 2905-2927, 1999). Inaddition, amplification, overexpression and/or activation of Akt havebeen detected in a number of human malignancies (Datta, S. R., et al.Genes Dev. 13: 2905-2927, 1999, Cheng, J. Q., and Nicosia, S. V. AKTsignal transduction pathway in oncogenesis. In Schwab D, editor.Encyclopedic Reference of Cancer. Berlin Heidelberg and New York:Springer; 2001. pp 35-7). Ectopic expression of Akt, especiallyconstitutively active Akt, induces cell survival and malignanttransformation whereas inhibition of Akt activity stimulates apoptosisin a range of mammalian cells (Datta, S. R., et al. Genes Dev. 13:2905-2927, 1999, Cheng, J. Q., and Nicosia, S. V. AKT signaltransduction pathway in oncogenesis. In Schwab D, editor. EncyclopedicReference of Cancer. Berlin Heidelberg and New York: Springer; 2001. pp35-7, Sun, M., et al. Am. J. Path., 159: 431-437, 2001, Cheng, J. Q., etal. Oncogene, 14: 2793-2801, 1997). Further, activation of Akt has beenshown to associate with tumor invasiveness and chemoresistance (West, K.A., et al. Drug Resist. Updat., 5: 234-248, 2002).

Activation of the Akt pathway plays a pivotal role in malignanttransformation and chemoresistance by inducing cell survival, growth,migration, and angiogenesis. The present invention provides methods todetermine levels of Akt kinase overexpression and/or hyperactivated andphosphorylated Akt kinase.

The Akt kinase can be any known Akt family kinase, or kinase relatedthereto, including, but not limited to Akt 1, Akt 2, Akt 3. The mRNA andamino acid sequences of human Akt1, Akt2, and Akt 3 are illustrated inFIGS. 6a -c, 7 a-d, and 8 a-c, respectively.

Diagnostic Assays Immunological Assays

In one embodiment, a method is provided for detecting the aberrantexpression of an Akt kinase in a cell in a mammal or in a biologicalsample from the mammal, by contacting cells, cell extracts or serum orother sample from the mammal or biological sample with animmunointeractive molecule specific for an Akt kinase or antigenicportion thereof and screening for the level of immunointeractivemolecule-Akt kinase complex formations and determining whether anelevated presence of the complex relative to a normal cell is present.

The immunointeractive molecule can be a molecule having specificity andbinding affinity for an Akt kinase or its antigenic parts or itshomologs or derivatives thereof. In one embodiment, theimmunointeractive molecule can be an immunglobulin molecule. In otherembodiments, the immunointeractive molecules can be an antibodyfragments, single chain antibodies, and/or deimmunized moleculesincluding humanized antibodies and T-cell associated antigen-bindingmolecules (TABMs). In one particular embodiment, the antibody can be amonoclonal antibody. In another particular embodiment, the antibody canbe a polyclonal antibody. The immunointeractive molecule can exhibitspecificity for an Akt kinase or more particularly an antigenicdeterminant or epitope on an Akt kinase. An antigenic determinant orepitope on an Akt kinase includes that part of the molecule to which animmune response is directed. The antigenic determinant or epitope can bea B-cell epitope or where appropriate a T-cell epitope. In oneembodiment, the antibody is a phosphor-ser 473 Akt antibody.

One embodiment of the present invention provides a method for diagnosingthe presence of cancer or cancer-like growth in a mammal, in whichaberrant Akt activity is present, by contacting cells or cell extractsfrom the mammal or a biological sample from the subject with an Aktkinase-binding effective amount of an antibody having specificity forthe Akt kinase or an antigenic determinant or epitope thereon and thenquantitatively or qualitatively determining the level of an Aktkinase-antibody complex wherein the presence of elevated levels of saidcomplex compared to a normal cell is determined.

Antibodies can be prepared by any of a number of means known to oneskilled in the art. For example, for the detection of human Akt kinase,antibodies can be generally but not necessarily derived from non-humananimals such as primates, livestock animals (e.g. sheep, cows, pigs,goats, horses), laboratory test animals (e.g. mice, rats, guinea pigs,rabbits) and/or companion animals (e.g. dogs, cats). Antibodies may alsobe recombinantly produced in prokaryotic or eukaryotic host cells.Generally, antibody based assays can be conducted in vitro on cell ortissue biopsies. However, if an antibody is suitably deimmunized or, inthe case of human use, humanized, then the antibody can be labeled with,for example, a nuclear tag, administered to a patient and the site ofnuclear label accumulation determined by radiological techniques. TheAkt kinase antibody can be a cancer targeting agent. Accordingly,another embodiment of the present invention provides deimmunized formsof the antibodies for use in cancer imaging in human and non-humanpatients.

In general, for the generation of antibodies to an Akt kinase, theenzyme is required to be extracted from a biological sample whether thisbe from animal including human tissue or from cell culture if producedby recombinant means. The Akt kinase can be separated from thebiological sample by any suitable means. For example, the separation maytake advantage of any one or more of the Akt kinases surface chargeproperties, size, density, biological activity and its affinity foranother entity (e.g. another protein or chemical compound to which itbinds or otherwise associates). Thus, for example, separation of the Aktkinase from the biological fluid can be achieved by any one or more ofultra-centrifugation, ion-exchange chromatography (e.g. anion exchangechromatography, cation exchange chromatography), electrophoresis (e.g.polyacrylamide gel electrophoresis, isoelectric focussing), sizeseparation (e.g., gel filtration, ultra-filtration) andaffinity-mediated separation (e.g. immunoaffinity separation including,but not limited to, magnetic bead separation such as Dynabead(trademark) separation, immunochromatography, immuno-precipitation). Theseparation of Akt kinase from the biological fluid can preserveconformational epitopes present on the kinase and, thus, suitably avoidstechniques that cause denaturation of the enzyme. In a furtherembodiment, the kinase can be separated from the biological fluid usingany one or more of affinity separation, gel filtration and/orultra-filtration.

Immunization and subsequent production of monoclonal antibodies can becarried out using standard protocols known in the art, such as, forexample, described by Kohler and Milstein (Kohler and Milstein, Nature256: 495-499, 1975; Kohler and Milstein, Eur. J. Immunol. 6(7): 511-519,1976), Coligan et al. (“Current Protocols in Immunology, John Wiley &Sons, Inc., 1991-1997) or Toyama et al. (Monoclonal Antibody, ExperimentManual”, published by Kodansha Scientific, 1987). Essentially, an animalis immunized with an Akt kinase-containing biological fluid or fractionthereof or a recombinant form of Akt kinase by standard methods toproduce antibody-producing cells, particularly antibody-producingsomatic cells (e.g. B lymphocytes). These cells can then be removed fromthe immunized animal for immortalization. In certain embodiment, afragment of an Akt kinase can be used to the generate antibodies. Thefragment can be associated with a carrier. The carrier can be anysubstance of typically high molecular weight to which a non- or poorlyimmunogenic substance (e.g. a hapten) is naturally or artificiallylinked to enhance its immunogenicity.

Immortalization of antibody-producing cells can be carried out usingmethods which are well-known in the art. For example, theimmortalization may be achieved by the transformation method usingEpstein-Barr virus (EBV) (Kozbor et al., Methods in Enzymology 121: 140,1986). In another embodiment, antibody-producing cells are immortalizedusing the cell fusion method (described in Coligan et al., 1991-1997,supra), which is widely employed for the production of monoclonalantibodies. In this method, somatic antibody-producing cells with thepotential to produce antibodies, particularly B cells, are fused with amyeloma cell line. These somatic cells may be derived from the lymphnodes, spleens and peripheral blood of primed animals, preferably rodentanimals such as mice and rats. In a particular embodiment, mice spleencells can be used. In other embodiments, rat, rabbit, sheep or goatcells can also be used. Specialized myeloma cell lines have beendeveloped from lymphocytic tumours for use in hybridoma-producing fusionprocedures (Kohler and Milstein, 1976, supra; Shulman et al., Nature276: 269-270, 1978; Volk et al., J. Virol. 42(1): 220-227, 1982). Manymyeloma cell lines can also be used for the production of fused cellhybrids, including, e.g. P3.times.63-Ag8, P3.times.63-AG8.653,P3/NS1-Ag4-1 (NS-1), Sp2/0-Ag14 and S194/5.XXO.Bu.1. The P3.times.63-Ag8and NS-1 cell lines have been described by Kohler and Milstein (1976,supra). Shulman et al. (1978, supra) developed the Sp2/0-Ag14 myelomaline. The S194/5.XXO.Bu.1 line was reported by Trowbridge (J. Exp. Med.148(1): 313-323, 1978). Methods for generating hybrids ofantibody-producing spleen or lymph node cells and myeloma cells usuallyinvolve mixing somatic cells with myeloma cells in a 10:1 proportion(although the proportion may vary from about 20:1 to about 1:1),respectively, in the presence of an agent or agents (chemical, viral orelectrical) that promotes the fusion of cell membranes. Fusion methodshave been described (Kohler and Milstein, 1975, supra; Kohler andMilstein, 1976, supra; Gefter et al., Somatic Cell Genet. 3: 231-236,1977; Volk et al., 1982, supra). The fusion-promoting agents used bythose investigators were Sendai virus and polyethylene glycol (PEG). Incertain embodiments, means to select the fused cell hybrids from theremaining unfused cells, particularly the unfused myeloma cells, areprovided. Generally, the selection of fused cell hybrids can beaccomplished by culturing the cells in media that support the growth ofhybridomas but prevent the growth of the unfused myeloma cells, whichnormally would go on dividing indefinitely. The somatic cells used inthe fusion do not maintain long-term viability in in vitro culture andhence do not pose a problem. Several weeks are required to selectivelyculture the fused cell hybrids. Early in this time period, it isnecessary to identify those hybrids which produce the desired antibody,so that they may subsequently be cloned and propagated. Generally,around 10% of the hybrids obtained produce the desired antibody,although a range of from about 1 to about 30% is not uncommon. Thedetection of antibody-producing hybrids can be achieved by any one ofseveral standard assay methods, including enzyme-linked immunoassay andradioimmunoassay techniques as, for example, described in Kennet et al.(Monoclonal Antibodies and Hybridomas: A New Dimension in BiologicalAnalyses, pp 376-384, Plenum Press, New York, 1980) and by FACS analysis(O'Reilly et al., Biotechniques 25: 824-830, 1998).

Once the desired fused cell hybrids have been selected and cloned intoindividual antibody-producing cell lines, each cell line may bepropagated in either of two standard ways. A suspension of the hybridomacells can be injected into a histocompatible animal. The injected animalwill then develop tumours that secrete the specific monoclonal antibodyproduced by the fused cell hybrid. The body fluids of the animal, suchas serum or ascites fluid, can be tapped to provide monoclonalantibodies in high concentration. Alternatively, the individual celllines may be propagated in vitro in laboratory culture vessels. Theculture medium containing high concentrations of a single specificmonoclonal antibody can be harvested by decantation, filtration orcentrifugation, and subsequently purified.

The cell lines can then be tested for their specificity to detect theAkt kinase of interest by any suitable immunodetection means. Forexample, cell lines can be aliquoted into a number of wells andincubated and the supernatant from each well is analyzed byenzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibodytechnique, or the like. The cell line(s) producing a monoclonal antibodycapable of recognizing the target LIM kinase but which does notrecognize non-target epitopes are identified and then directly culturedin vitro or injected into a histocompatible animal to form tumours andto produce, collect and purify the required antibodies.

The present invention provides, therefore, a method of detecting in asample an Akt kinase or fragment, variant or derivative thereofcomprising contacting the sample with an antibody or fragment orderivative thereof and detecting the level of a complex containing theantibody and Akt kinase or fragment, variant or derivative thereofcompared to normal controls wherein elevated levels of Akt kinase isdetermined. Any suitable technique for determining formation of thecomplex may be used. For example, an antibody according to theinvention, having a reporter molecule associated therewith, may beutilized in immunoassays. Such immunoassays include but are not limitedto radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs)immunochromatographic techniques (ICTs), and Western blotting which arewell known to those of skill in the art. Immunoassays can also includecompetitive assays. The present invention encompasses qualitative andquantitative immunoassays.

Suitable immunoassay techniques are described, for example, in U.S. Pat.Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-siteand two-site assays of the non-competitive types, as well as thetraditional competitive binding assays. These assays also include directbinding of a labeled antigen-binding molecule to a target antigen.

The invention further provides methods for quantifying Akt proteinexpression and activation levels in cells or tissue samples obtainedfrom an animal, such as a human cancer patient or an individualsuspected of having cancer. In one embodiment, the invention providesmethods for quantifying Akt protein expression or activation levelsusing an imaging system quantitatively. The imaging system can be usedto receive, enhance, and process images of cells or tissue samples, thathave been stained with AKT protein-specific stains, in order todetermine the amount or activation level of AKT protein expressed in thecells or tissue samples from such an animal. In embodiments of themethods of the invention, a calibration curve of AKT1 and AKT2 proteinexpression can be generated for at least two cell lines expressingdiffering amounts of AKT protein. The calibration curve can then used toquantitatively determine the amount of AKT protein that is expressed ina cell or tissue sample. Analogous calibration curves can be made foractivated AKT proteins using reagents specific for the activationfeatures. It can also be used to determine changes in amounts andactivation state of AKT before and after clinical cancer treatment.

In one particular embodiment of the methods of the invention, AKTprotein expression in a cell or tissue sample can be quantified using anenzyme-linked immunoabsorbent assay (ELISA) to determine the amount ofAKT protein in a sample. Such methods are described, for example, inU.S. Patent Publication No. 2002/0015974.

In other embodiments enzyme immunoassays can be used to detect the Aktkinase. In such assays, an enzyme is conjugated to the second antibody,generally by means of glutaraldehyde or periodate. The substrates to beused with the specific enzymes are generally chosen for the productionof, upon hydrolysis by the corresponding enzyme, a detectable colourchange. It is also possible to employ fluorogenic substrates, whichyield a fluorescent product rather than the chromogenic substrates. Theenzyme-labeled antibody can be added to the first antibody-antigencomplex, allowed to bind, and then the excess reagent washed away. Asolution containing the appropriate substrate can then be added to thecomplex of antibody-antigen-antibody. The substrate can react with theenzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of antigenwhich was present in the sample. Alternately, fluorescent compounds,such as fluorescein, rhodamine and the lanthanide, europium (EU), can bechemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic colour visually detectable with a lightmicroscope. The fluorescent-labeled antibody is allowed to bind to thefirst antibody-antigen complex. After washing off the unbound reagent,the remaining tertiary complex is then exposed to light of anappropriate wavelength. The fluorescence observed indicates the presenceof the antigen of interest. Immunofluorometric assays (IFMA) are wellestablished in the art and are particularly useful for the presentmethod. However, other reporter molecules, such as radioisotope,chemiluminescent or bioluminescent molecules can also be employed.

In a particular embodiment, antibodies to Akt kinase can also be used inELISA-mediated detection of Akt kinase especially in serum or othercirculatory fluid. This can be accomplished by immobilizing anti-Aktkinase antibodies to a solid support and contacting these with abiological extract such as serum, blood, lymph or other bodily fluid,cell extract or cell biopsy. Labeled anti-Akt kinase antibodies can thenbe used to detect immobilized Akt kinase. This assay can be varied inany number of ways and all variations are encompassed by the presentinvention and known to one skilled in the art. This approach can enablerapid detection and quantitation of Akt kinase levels using, forexample, a serum-based assay.

In one embodiment, an Akt Elisa assay kit may be used in the presentinvention. For example, a Cellular Activation of Signaling ELISA kit forAkt S473 from SuperArray Bioscience can be utilized in the presentinvention. In one embodiment, the antibody can be an anti-pan antibodythat recognizes Akt S473. Elisa assay kit containing an anti-Aktantibody and additional reagents, including, but not limited to, washingbuffer, antibody dilution buffer, blocking buffer, cell stainingsolution, developing solution, stop solution, secondary antibodies, anddistilled water.

Nucleotide Detection

In another embodiment, a method to detect Akt kinases is provided bydetecting the level of expression in a cell of a polynucleotide encodingan Akt kinase. Expression of the polynucleotide can be determined usingany suitable technique known to one skilled in the art. In oneembodiment, a labeled polynucleotide encoding an Akt kinase can beutilized as a probe in a Northern blot of an RNA extract obtained fromthe cell. In other embodiments, a nucleic acid extract from an animalcan be utilized in concert with oligonucleotide primers corresponding tosense and antisense sequences of a polynucleotide encoding the kinase,or flanking sequences thereof, in a nucleic acid amplification reactionsuch as RT PCR. A variety of automated solid-phase detection techniquesare also available to one skilled in the art, for example, as describedby Fodor et al. (Science 251: 767-777, 1991) and Kazal et al. (NatureMedicine 2: 753-759, 1996).

In other embodiments, methods are provided to detect akt kinase encodingRNA transcripts. The RNA can be isolated from a cellular samplesuspected of containing Akt kinase RNA, e.g. total RNA isolated fromhuman cancer tissue. RNA can be isolated by methods known in the art,e.g. using TRIZOL reagent (GIBCO-BRL/Life Technologies, Gaithersburg,Md.). Oligo-dT, or random-sequence oligonucleotides, as well assequence-specific oligonucleotides can be employed as a primer in areverse transcriptase reaction to prepare first-strand cDNAs from theisolated RNA. Resultant first-strand cDNAs can then amplified withsequence-specific oligonucleotides in PCR reactions to yield anamplified product.

Polymerase chain reaction or “PCR” refers to a procedure or technique inwhich amounts of a preselected fragment of nucleic acid, RNA and/or DNA,are amplified as described, for example, in U.S. Pat. No. 4,683,195.Generally, sequence information from the ends of the region of interestor beyond is employed to design oligonucleotide primers. These primerswill be identical or similar in sequence to opposite strands of thetemplate to be amplified. PCR can be used to amplify specific RNAsequences and cDNA transcribed from total cellular RNA. See generallyMullis et al. (Quant. Biol. 51: 263, 1987; Erlich, eds., PCR Technology,Stockton Press, NY, 1989). Thus, amplification of specific nucleic acidsequences by PCR relies upon oligonucleotides or “primers” havingconserved nucleotide sequences wherein the conserved sequences arededuced from alignments of related gene or protein sequences, e.g. asequence comparison of mammalian Akt kinase genes. For example, oneprimer is prepared which is predicted to anneal to the antisense strandand another primer prepared which is predicted to anneal to the sensestrand of a cDNA molecule which encodes a Akt kinase. To detect theamplified product, the reaction mixture is typically subjected toagarose gel electrophoresis or other convenient separation technique andthe relative presence of the Akt kinase specific amplified DNA detected.For example, Akt kinase amplified DNA may be detected using Southernhybridization with a specific oligonucleotide probe or comparing itselectrophoretic mobility with DNA standards of known molecular weight.Isolation, purification and characterization of the amplified Akt kinaseDNA can be accomplished by excising or eluting the fragment from the gel(for example, see references Lawn et al., Nucleic Acids Res. 2: 6103,1981; Goeddel et al., Nucleic cids Res. 8: 4057-1980), cloning theamplified product into a cloning site of a suitable vector, such as thepCRII vector (Invitrogen), sequencing the cloned insert and comparingthe DNA sequence to the known sequence of LIM kinase. The relativeamounts of LIM kinase mRNA and cDNA can then be determined.

In one embodiment, real-time PCR can be used to determinetranscriptional levels of Akt nucleotides. Determination oftranscriptional activity also includes a measure of potentialtranslational activity based on available mRNA transcripts. Real-timePCR as well as other PCR procedures use a number of chemistries fordetection of PCR product including the binding of DNA bindingfluorophores, the 5′ endonuclease, adjacent liner and hairpinoligoprobes and the self-fluorescing amplicons. These chemistries andreal-time PCR in general are discussed, for example, in Mackay et al.,Nucleic Acids Res 30(6): 1292-1305, 2002; Walker, J. Biochem. Mol.Toxicology 15(3): 121-127, 2001; Lewis et al., J. Pathol. 195: 66-71,2001.

In an alternate embodiment, the aberrant expression of Akt can beidentified by contacting a nucleotide sequences isolated from abiological sample with an oligonucleotide probe having a sequencecomplementary to an Akt sequences selected from the nucleotide sequencesof FIGS. 6a -c, 7 a-d, or 8 a-c, or fragment thereof, and then detectingthe sequence by hybridizing the probe to the sequence, and comparing theresults to a normal sample. The hybridization of the probe to thebiological sample can be detected by labeling the probe using anydetectable agent. The probe can be labeled for example, with aradioisotope, or with biotin, fluorescent dye, electron-dense reagent,enzyme, hapten or protein for which antibodies are available. Thedetectable label can be assayed by any desired means, includingspectroscopic, photochemical, biochemical, immunochemical,radioisotopic, or chemical means. The probe can also be detected usingtechniques such as an oligomer restriction technique, a dot blot assay,a reverse dot blot assay, a line probe assay, and a 5′ nuclease assay.Alternatively, the probe can be detected using any of the generallyapplicable DNA array technologies, including macroarray, microarray andDNA microchip technologies. The oligonucleotide probe typically includesapproximately at least 14, 15, 16, 18, 20, 25 or 28 nucleotides thathybridize to the nucleotides selected from FIGS. 6a -c, 7 a-d, and 8a-c, or a fragment thereof. It is generally not preferred to use a probethat is greater than approximately 25 or 28 nucleotides in length. Theoligonucleotide probe is designed to identify an Akt nucleotidesequence.

Kinase Assays

The activity of the Akt kinases can be measured using any suitablekinase assay known in the art. For example, and not by way oflimitation, the methods described in Hogg et al (Oncogene 1994 9:98-96),Mills et al (J. Biol. Chem. 1992 267:16000-006) and Tomizawa et al 2001(FEBS Lett. 2001 492: 221-7), Schmandt et al, (J. Immunol. 1994,152:96-105) can be used. Further serine, threonine and tyrosine kinaseassays are described in Ausubel et al. (Short Protocols in MolecularBiology, 1999, unit 17.6).

Akt kinase assays can generally use an Akt polypeptide, a labeled donorsubstrate, and a receptor substrate that is either specific ornon-specific for Akt. In such assays Akt transfers a labeled moiety fromthe donor substrate to the receptor substrate, and kinase activity ismeasured by the amount of labeled moiety transferred from the donorsubstrate to the receptor substrate. Akt polypeptide can be producedusing various expression systems, can be purified from cells, can be inthe form of a cleaved or uncleaved recombinant fusion protein and/or canhave non-Akt polypeptide sequences, for example a His tag or.beta.-galactosidase at its N- or C-terminus. Akt activity can beassayed in cancerous cells lines if the cancerous cell lines are used asa source of the Akt to be assayed. Suitable donor substrates for Aktassays include any molecule that is susceptible to dephosphorylation byAkt., such as, for example include .gamma.-labeled ATP and ATP analogs,wherein the label is ³³P, ³²P, ³⁵S or any other radioactive isotope or asuitable fluorescent marker. Suitable recipient substrates for Aktassays include any polypeptide or other molecule that is susceptible tophosphorylation by Akt. Recipient substrates can be derived fromfragments of in vivo targets of Akt. Recipient substrates fragments canbe 8 to 50 amino acids in length, usually 10 to 30 amino acids andparticularly of about 10, 12, 15, 18, 20 and 25 amino acids in length.Further recipient substrates can be determined empirically using a setof different polypeptides or other molecules. Targets of Recipientsubstrates for TTK can be capable of being purified from othercomponents of the reaction once the reaction has been performed. Thispurification is usually done through a molecular interaction, where therecipient substrates is biotinylated and purified through itsinteraction with streptavidin, or a specific antibody is available thatcan specifically recognize the recipient substrates. The reaction can beperformed in a variety of conditions, such as on a solid support, in agel, in solution or in living cells. The choice of detection methodsdepends on type of label used for the donor molecule and may include,for example, measurement of incorporated radiation or fluorescence byautoradiography, scintillation, scanning or fluorography.

IV. Methods of Treatment

The compounds and pharmaceutical compositions provided herein can beused in the treatment of esophogeal adenocarcinoma and other disordersassociated with abnormal cell proliferation. In one embodiment, thecompounds of the present invention can be used to treat esophogealadenocarcinoma.

Drug Resistant Tumors or Cancers

The invention provides compounds that can be used to treat drugresistant esophogeal adenocarcinoma. In one embodiment, the compound,such as TCN, TCN-P or a related compound as disclosed herein, can beco-administered with a second drug.

Multidrug resistance (MDR) occurs in human cancers and can be asignificant obstacle to the success of chemotherapy. Multidrugresistance is a phenomenon whereby tumor cells in vitro that have beenexposed to one cytotoxic agent develop cross-resistance to a range ofstructurally and functionally unrelated compounds. In addition, MDR canoccur intrinsically in some cancers without previous exposure tochemotherapy agents. Thus, in one embodiment, the present inventionprovides methods for the treatment of a patient with a drug resistantcancer, for example, multidrug resistant cancer, by administration ofTCN, TCN-P or a related compound as disclosed herein. In certainembodiments, TCN, TCN-P and related compounds can be used to treatcancers that are resistant to taxol, rapamycin, tamoxifen, cisplatin,and/or gefitinib (iressa).

In one embodiment, TCN, TCN-P or a related compound as disclosed hereincan be used for the treatment of drug resistent cancers of the colon,bone, kidney, adrenal, pancreas, liver and/or any other cancer known inthe art or described herein.

Combination Therapy

In one aspect of the present invention, the compounds and compositionsdisclosed herein can be combined with at least one additionalchemotherapeutic agent. The additional agents can be administered incombination or alternation with the compounds disclosed herein. Thedrugs can form part of the same composition, or be provided as aseparate composition for administration at the same time or a differenttime.

In one embodiment, compounds disclosed herein can be combined withantiangiogenic agents to enhance their effectiveness, or combined withother antiangiogenic agents and administered together with othercytotoxic agents. In another embodiment, the compounds and compositions,when used in the treatment of solid tumors, can be administered with theagents selected from, but not limited to IL-12, retinoids, interferons,angiostatin, endostatin, thalidomide, thrombospondin-1,thrombospondin-2, captopryl, anti-neoplastic agents such as alphainterferon, COMP (cyclophosphamide, vincristine, methotrexate andprednisone), etoposide, mBACOD (methortrexate, bleomycin, doxorubicin,cyclophosphamide, vincristine and dexamethasone), PRO-MACE/MOPP(prednisone, methotrexate (w/leucovin rescue), doxorubicin,cyclophosphamide, taxol, etoposide/mechlorethamine, vincristine,prednisone and procarbazine), vincristine, vinblastine, angioinhibins,TNP-470, pentosan polysulfate, platelet factor 4, angiostatin, LM-609,SU-101, CM-101, Techgalan, thalidomide, SP-PG and radiation. In furtherembodiments, the compounds and compositions disclosed herein can beadministered in combination or alternation with, for example, drugs withantimitotic effects, such as those which target cytoskeletal elements,including microtubule modulators such as taxane drugs (such as taxol,paclitaxel, taxotere, docetaxel), podophylotoxins or vinca alkaloids(vincristine, vinblastine); antimetabolite drugs (such as5-fluorouracil, cytarabine, gemcitabine, purine analogues such aspentostatin, methotrexate); alkylating agents or nitrogen mustards (suchas nitrosoureas, cyclophosphamide or ifosphamide); drugs which targetDNA such as the antracycline drugs adriamycin, doxorubicin,pharmorubicin or epirubicin; drugs which target topoisomerases such asetoposide; hormones and hormone agonists or antagonists such asestrogens, antiestrogens (tamoxifen and related compounds) andandrogens, flutamide, leuprorelin, goserelin, cyprotrone or octreotide;drugs which target signal transduction in tumour cells includingantibody derivatives such as herceptin; alkylating drugs such asplatinum drugs (cis-platin, carbonplatin, oxaliplatin, paraplatin) ornitrosoureas; drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors; gene therapy and antisense agents;antibody therapeutics; other bioactive compounds of marine origin,notably the didemnins such as aplidine; steroid analogues, in particulardexamethasone; anti-inflammatory drugs, including nonsteroidal agents(such as acetaminophen or ibuprofen) or steroids and their derivativesin particular dexamethasone; anti-emetic drugs, including 5HT-3inhibitors (such as gramisetron or ondasetron), and steroids and theirderivatives in particular dexamethasone. In still further embodiments,the compounds and compositions can be used in combination or alternationwith the chemotherapeutic agents disclosed below in Table 1.

TABLE 1 Chemotherapeutic Agents 13-cis-Retinoic Acid Neosar2-Amino-6-Mercaptopurine Neulasta 2-CdA Neumega 2-ChlorodeoxyadenosineNeupogen 5-fluorouracil Nilandron 5-FU Nilutamide 6-TG Nitrogen Mustard6-Thioguanine Novaldex 6-Mercaptopurine Novantrone 6-MP OctreotideAccutane Octreotide acetate Actinomycin-D Oncospar Adriamycin OncovinAdrucil Ontak Agrylin Onxal Ala-Cort Oprevelkin Aldesleukin OrapredAlemtuzumab Orasone Alitretinoin Oxaliplatin Alkaban-AQ PaclitaxelAlkeran Pamidronate All-transretinoic acid Panretin Alpha interferonParaplatin Altretamine Pediapred Amethopterin PEG Interferon AmifostinePegaspargase Aminoglutethimide Pegfilgrastim Anagrelide PEG-INTRONAnandron PEG-L-asparaginase Anastrozole Phenylalanine MustardArabinosylcytosine Platinol Ara-C Platinol-AQ Aranesp PrednisoloneAredia Prednisone Arimidex Prelone Aromasin Procarbazine Arsenictrioxide PROCRIT Asparaginase Proleukin ATRA Prolifeprospan 20 withCarmustine implant Avastin Purinethol BCG Raloxifene BCNU RheumatrexBevacizumab Rituxan Bexarotene Rituximab Bicalutamide Roveron-A(interferon alfa-2a) BiCNU Rubex Blenoxane Rubidomycin hydrochlorideBleomycin Sandostatin Bortezomib Sandostatin LAR Busulfan SargramostimBusulfex Solu-Cortef C225 Solu-Medrol Calcium Leucovorin STI-571 CampathStreptozocin Camptosar Tamoxifen Camptothecin-11 Targretin CapecitabineTaxol Carac Taxotere Carboplatin Temodar Carmustine TemozolomideCarmustine wafer Teniposide Casodex TESPA CCNU Thalidomide CDDP ThalomidCeeNU TheraCys Cerubidine Thioguanine cetuximab Thioguanine TabloidChlorambucil Thiophosphoamide Cisplatin Thioplex Citrovorum FactorThiotepa Cladribine TICE Cortisone Toposar Cosmegen Topotecan CPT-11Toremifene Cyclophosphamide Trastuzumab Cytadren Tretinoin CytarabineTrexall Cytarabine liposomal Trisenox Cytosar-U TSPA Cytoxan VCRDacarbazine Velban Dactinomycin Velcade Darbepoetin alfa VePesidDaunomycin Vesanoid Daunorubicin Viadur Daunorubicin hydrochlorideVinblastine Daunorubicin liposomal Vinblastine Sulfate DaunoXomeVincasar Pfs Decadron Vincristine Delta-Cortef Vinorelbine DeltasoneVinorelbine tartrate Denileukin diftitox VLB DepoCyt VP-16 DexamethasoneVumon Dexamethasone acetate Xeloda dexamethasone sodium Zanosarphosphate Zevalin Dexasone Zinecard Dexrazoxane Zoladex DHAD Zoledronicacid DIC Zometa Diodex Gliadel wafer Docetaxel Glivec Doxil GM-CSFDoxorubicin Goserelin Doxorubicin liposomal granulocyte - colonystimulating factor Droxia Granulocyte macrophage colony stimulatingfactor DTIC Halotestin DTIC-Dome Herceptin Duralone Hexadrol EfudexHexalen Eligard Hexamethylmelamine Ellence HMM Eloxatin Hycamtin ElsparHydrea Emcyt Hydrocort Acetate Epirubicin Hydrocortisone Epoetin alfaHydrocortisone sodium phosphate Erbitux Hydrocortisone sodium succinateErwinia L-asparaginase Hydrocortone phosphate Estramustine HydroxyureaEthyol Ibritumomab Etopophos Ibritumomab Tiuxetan Etoposide IdamycinEtoposide phosphate Idarubicin Eulexin Ifex Evista IFN-alpha ExemestaneIfosfamide Fareston IL-2 Faslodex IL-11 Femara Imatinib mesylateFilgrastim Imidazole Carboxamide Floxuridine Interferon alfa FludaraInterferon Alfa-2b (PEG conjugate) Fludarabine Interleukin-2 FluoroplexInterleukin-11 Fluorouracil Intron A (interferon alfa-2b) Fluorouracil(cream) Leucovorin Fluoxymesterone Leukeran Flutamide Leukine FolinicAcid Leuprolide FUDR Leurocristine Fulvestrant Leustatin G-CSF LiposomalAra-C Gefitinib Liquid Pred Gemcitabine Lomustine Gemtuzumab ozogamicinL-PAM Gemzar L-Sarcolysin Gleevec Meticorten Lupron Mitomycin LupronDepot Mitomycin-C Matulane Mitoxantrone Maxidex M-PrednisolMechlorethamine MTC Mechlorethamine MTX Hydrochlorine MustargenMedralone Mustine Medrol Mutamycin Megace Myleran Megestrol IressaMegestrol Acetate Irinotecan Melphalan Isotretinoin MercaptopurineKidrolase Mesna Lanacort Mesnex L-asparaginase Methotrexate LCRMethotrexate Sodium Methylprednisolone Mylocel Letrozole

In certain embodiments, interferons (IFNs) can be used in combinationswith the compounds of the present invention. Suitable intereferonsinclude: interferon alpha-2a, interferon alpha-2b, pegylated interferonalpha, including interferon alpha-2a and interferon alpha 2b, interferonbeta, interferon gamma, interferon tau, interferon omega, INFERGEN(interferon alphacon-1) by InterMune, OMNIFERON (natural interferon) byViragen, ALBUFERON by Human Genome Sciences, REBIF (interferon beta-1a)by Ares-Serono, Omega Interferon by BioMedicine, Oral Interferon Alphaby Amarillo Biosciences, and interferon gamma, interferon tau, and/orinterferon gamma-1b by InterMune.

In one embodiment TCN, TCN-P or a related compound as disclosed hereincan be used in combination or alternation with additionalchemotherapeutic agents, such as those described herein or in Table 3,for the treatment of drug resistant cancer, for example multiple drugresistant cancer. Drug resistent cancers can include cancers of thecolon, bone, kidney, adrenal, pancreas, liver and/or any other cancerknown in the art or described herein. In one embodiment, the additionalchemotherapeutic agent can be a P-glycoprotein inhibitor. In certainnon-limiting embodiments, the P-glycoprotein inhibitor can be selectedfrom the following drugs: verapamil, cyclosporin (such as cyclosporinA), tamoxifen, calmodulin antagonists, dexverapamil, dexniguldipine,valspodar (PSC 833), biricodar (VX-710), tariquidar (XR9576), zosuquidar(LY335979), laniquidar (R101933), and/or ONT-093.

V. Pharmaceutical Compositions

Pharmaceutical carriers suitable for administration of the compoundsprovided herein include any such carriers known to those skilled in theart to be suitable for the particular mode of administration. Thecompounds may be formulated as the sole pharmaceutically activeingredient in the composition or may be combined with other activeingredients.

Compositions comprising the compounds disclosed herein may be suitablefor oral, rectal, nasal, topical (including buccal and sublingual),vaginal, or parenteral (including subcutaneous, intramuscular,subcutaneous, intravenous, intradermal, intraocular, intratracheal,intracisternal, intraperitoneal, and epidural) administration.

The compositions may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association one or morecompositions of the present invention and one or more pharmaceuticalcarriers or excipients.

The compounds can be formulated into suitable pharmaceuticalpreparations such as solutions, suspensions, tablets, dispersibletablets, pills, capsules, powders, sustained release formulations orelixirs, for oral administration or in sterile solutions or suspensionsfor parenteral administration, as well as transdermal patch preparationand dry powder inhalers. In one embodiment, the compounds describedabove are formulated into pharmaceutical compositions using techniquesand procedures well known in the art (see, e.g., Ansel Introduction toPharmaceutical Dosage Forms, Fourth Edition 1985, 126).

In the compositions, effective concentrations of one or more compoundsor pharmaceutically acceptable derivatives thereof may be mixed with oneor more suitable pharmaceutical carriers. The compounds may bederivatized as the corresponding salts, esters, enol ethers or esters,acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases,solvates, hydrates or prodrugs prior to formulation. The concentrationsof the compounds in the compositions are effective for delivery of anamount, upon administration, that treats, prevents, or ameliorates oneor more of the symptoms of the target disease or disorder. In oneembodiment, the compositions are formulated for single dosageadministration. To formulate a composition, the weight fraction ofcompound is dissolved, suspended, dispersed or otherwise mixed in aselected carrier at an effective concentration such that the treatedcondition is relieved, prevented, or one or more symptoms areameliorated.

Compositions suitable for oral administration may be presented asdiscrete units such as, but not limited to, tablets, caplets, pills ordragees capsules, or cachets, each containing a predetermined amount ofone or more of the compositions; as a powder or granules; as a solutionor a suspension in an aqueous liquid or a non-aqueous liquid; or as anoil-in-water liquid emulsion or a water-in-oil emulsion or as a bolus,etc.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecompound as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents, preservatives, flavoring agents, and thelike, for example, acetate, sodium citrate, cyclodextrine derivatives,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, and other such agents. Methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 15th Edition, 1975.

Compositions of the present invention suitable for topicaladministration in the mouth include for example, lozenges, having theingredients in a flavored basis, usually sucrose and acacia ortragacanth; pastilles, having one or more of the compositions of thepresent invention in an inert basis such as gelatin and glycerin, orsucrose and acacia; and mouthwashes, having one or more of thecompositions of the present invention administered in a suitable liquidcarrier.

The tablets, pills, capsules, troches and the like can contain one ormore of the following ingredients, or compounds of a similar nature: abinder; a lubricant; a diluent; a glidant; a disintegrating agent; acoloring agent; a sweetening agent; a flavoring agent; a wetting agent;an emetic coating; and a film coating. Examples of binders includemicrocrystalline cellulose, gum tragacanth, glucose solution, acaciamucilage, gelatin solution, molasses, polvinylpyrrolidine, povidone,crospovidones, sucrose and starch paste. Lubricants include talc,starch, magnesium or calcium stearate, lycopodium and stearic acid.Diluents include, for example, lactose, sucrose, starch, kaolin, salt,mannitol and dicalcium phosphate. Glidants include, but are not limitedto, colloidal silicon dioxide. Disintegrating agents includecrosscarmellose sodium, sodium starch glycolate, alginic acid, cornstarch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Coloring agents include, for example, any of theapproved certified water soluble FD and C dyes, mixtures thereof; andwater insoluble FD and C dyes suspended on alumina hydrate. Sweeteningagents include sucrose, lactose, mannitol and artificial sweeteningagents such as saccharin, and any number of spray dried flavors.Flavoring agents include natural flavors extracted from plants such asfruits and synthetic blends of compounds which produce a pleasantsensation, such as, but not limited to peppermint and methyl salicylate.Wetting agents include propylene glycol monostearate, sorbitanmonooleate, diethylene glycol monolaurate and polyoxyethylene lauralether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

Compositions suitable for topical administration to the skin may bepresented as ointments, creams, gels, and pastes, having one or more ofthe compositions administered in a pharmaceutical acceptable carrier.

Compositions for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Compositions suitable for nasal administration, when the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is taken, (i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose). Whenthe carrier is a liquid (for example, a nasal spray or as nasal drops),one or more of the compositions can be admixed in an aqueous or oilysolution, and inhaled or sprayed into the nasal passage.

Compositions suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining one or more of the compositions and appropriate carriers.

Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats, and solutes which render the formulationisotonic with the blood of the intended recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents andthickening agents. The compositions may be presented in unit-dose ormulti-dose containers, for example, sealed ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules, andtablets of the kind previously described above.

Pharmaceutical organic or inorganic solid or liquid carrier mediasuitable for enteral or parenteral administration can be used tofabricate the compositions. Gelatin, lactose, starch, magnesiumstearate, talc, vegetable and animal fats and oils, gum, polyalkyleneglycol, water, or other known carriers may all be suitable as carriermedia.

Compositions may be used as the active ingredient in combination withone or more pharmaceutically acceptable carrier mediums and/orexcipients. As used herein, “pharmaceutically acceptable carrier medium”includes any and all carriers, solvents, diluents, or other liquidvehicles, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants, adjuvants, vehicles, delivery systems, disintegrants,absorbents, preservatives, surfactants, colorants, flavorants, orsweeteners and the like, as suited to the particular dosage formdesired.

Additionally, the compositions may be combined with pharmaceuticallyacceptable excipients, and, optionally, sustained-release matrices, suchas biodegradable polymers, to form therapeutic compositions. A“pharmaceutically acceptable excipient” includes a non-toxic solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type.

It will be understood, however, that the total daily usage of thecompositions will be decided by the attending physician within the scopeof sound medical judgment. The specific therapeutically effective doselevel for any particular host will depend upon a variety of factors,including for example, the disorder being treated and the severity ofthe disorder; activity of the specific composition employed; thespecific composition employed, the age, body weight, general health, sexand diet of the patient; the time of administration; route ofadministration; rate of excretion of the specific compound employed; theduration of the treatment; drugs used in combination or coincidentialwith the specific composition employed; and like factors well known inthe medical arts. For example, it is well within the skill of the art tostart doses of the composition at levels lower than those required toachieve the desired therapeutic effect and to gradually increase thedosage until the desired effect is achieved.

Compositions are preferably formulated in dosage unit form for ease ofadministration and uniformity of dosage. “Dosage unit form” as usedherein refers to a physically discrete unit of the compositionappropriate for the host to be treated. Each dosage should contain thequantity of composition calculated to produce the desired therapeuticaffect either as such, or in association with the selectedpharmaceutical carrier medium.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, or an appropriate fraction thereof, of theadministered ingredient. For example, approximately 1-5 mg per day of acompound disclosed herein can reduce the volume of a solid tumor inmice.

The dosage will depend on host factors such as weight, age, surfacearea, metabolism, tissue distribution, absorption rate and excretionrate. In one embodiment, approximately 0.5 to 7 grams per day of acompound disclosed herein may be administered to humans. Optionally,approximately 1 to 4 grams per day of the compound can be administeredto humans. In certain embodiments 0.001-5 mg/day is administered to ahuman. The therapeutically effective dose level will depend on manyfactors as noted above. In addition, it is well within the skill of theart to start doses of the composition at relatively low levels, andincrease the dosage until the desired effect is achieved.

Compositions comprising a compound disclosed herein may be used with asustained-release matrix, which can be made of materials, usuallypolymers, which are degradable by enzymatic or acid-based hydrolysis orby dissolution. Once inserted into the body, the matrix is acted upon byenzymes and body fluids. A sustained-release matrix for example ischosen from biocompatible materials such as liposomes, polylactides(polylactic acid), polyglycolide (polymer of glycolic acid), polylactideco-glycolide (copolymers of lactic acid and glycolic acid),polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxcylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferredbiodegradable matrix is a matrix of one of either polylactide,polyglycolide, or polylactide co-glycolide (co-polymers of lactic acidand glycolic acid).

The compounds may also be administered in the form of liposomes. As isknown in the art, liposomes are generally derived from phospholipids orother lipid substances. Liposomes are formed by mono- or multi-lamellarhydrated liquid crystals that are dispersed in an aqueous medium. Anynon-toxic, physiologically-acceptable and metabolizable lipid capable offorming liposomes can be used. The liposome can contain, in addition toone or more compositions of the present invention, stabilizers,preservatives, excipients, and the like. Examples of lipids are thephospholipids and the phosphatidyl cholines (lecithins), both naturaland synthetic. Methods to form liposomes are known in the art.

The compounds may be formulated as aerosols for application, such as byinhalation. These formulations for administration to the respiratorytract can be in the form of an aerosol or solution for a nebulizer, oras a microfine powder for insufflation, alone or in combination with aninert carrier such as lactose. In such a case, the particles of theformulation will, in one embodiment, have diameters of less than 50microns, in one embodiment less than 10 microns.

Compositions comprising the compounds disclosed herein may be used incombination with other compositions and/or procedures for the treatmentof the conditions described above. For example, a tumor may be treatedconventionally with surgery, radiation, or chemotherapy combined withone or more compositions of the present invention and then one or morecompositions of the present invention may be subsequently administeredto the patient to extend the dormancy of micrometastases and tostabilize, inhibit, or reduce the growth of any residual primary tumor.

Additional Embodiments

The pharmaceutical compositions of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in a number of sourceswhich are well known and readily available to those skilled in the art.For example, Remington's Pharmaceutical Sciences (Martin E W [1995]Easton Pa., Mack Publishing Company, 19^(th) ed.) describes formulationswhich can be used in connection with the subject invention. Formulationssuitable for administration include, for example, aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient; and aqueous and nonaqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

The methods of the present invention, for example, for inhibiting thegrowth of a cancerous cell, can be advantageously combined with at leastone additional therapeutic method, including but not limited tochemotherapy, radiation therapy, therapy that selectively inhibits Rasoncogenic signaling, or any other therapy known to those of skill in theart of the treatment and management of cancer, such as administration ofan anti-cancer agent.

Administration of API-2 (triciribine) as a salt may be carried out.Examples of pharmaceutically acceptable salts are organic acid additionsalts formed with acids which form a physiological acceptable anion, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, andalpha-glycerophosphate. Suitable inorganic salts may also be formed,including hydrochloride, sulfate, nitrate, bicarbonate, and carbonatesalts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The compounds of the present invention can be formulated aspharmaceutical compositions and administered to a subject, such as ahuman or veterinary patient, in a variety of forms adapted to the chosenroute of administration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the compounds of the present invention may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle (i.e., carrier) such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the compounds may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% ofactive agent. The percentage of the compositions and preparations may,of course, be varied and may conveniently be between about 2 to about60% of the weight of a given unit dosage form. The amount of the activecompound in such therapeutically useful compositions is such that aneffective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the compounds of the invention, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, thecompounds of the invention may be incorporated into sustained-releasepreparations and devices.

The active agent (i.e., API-2 or pharmaceutically acceptable saltsthereof) may also be administered intravenously or intraperitoneally byinfusion or injection. Solutions of the active agent or its salts can beprepared in water, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating compounds ofthe invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, the compounds of the invention may beapplied in pure-form, i.e., when they are liquids. However, it willgenerally be desirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds of the invention can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver the compounds of the invention to the skinare disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Woltzman(U.S. Pat. No. 4,820,508).

Useful dosages of the pharmaceutical compositions of the presentinvention can be determined by comparing their in vitro activity, and invivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

In one non-limiting embodiment, the concentration of the active agent ina liquid composition, such as a lotion, can be from about 0.1-25 wt-%,or from about 0.5-10 wt.-%. In one embodiment, the concentration in asemi-solid or solid composition such as a gel or a powder can be about0.1-5 wt.-%, preferably about 0.5-2.5 wt.-%. In one embodiment, singledosages for injection, infusion or ingestion will generally vary between5-1500 mg, and may be administered, i.e., 1-3 times daily, to yieldlevels of about 0.1-50 mg/kg, for adults. A non-limiting dosage of thepresent invention can be between 7.5 to 45 mg per clay, administeredorally, with appropriate adjustment for the body weight of anindividual.

Accordingly, the present invention includes a pharmaceutical compositioncomprising API-2, as described herein, or pharmaceutically acceptablesalts thereof, in combination with a pharmaceutically acceptablecarrier. Pharmaceutical compositions adapted for oral, topical orparenteral administration, comprising an amount of API-2, or apharmaceutically acceptable salt thereof, constitute a preferredembodiment of the invention. The dose administered to a subject,particularly a human, in the context of the present invention should besufficient to effect a therapeutic response in the patient over areasonable time frame. One skilled in the art will recognize that dosagewill depend upon a variety of factors including the condition of theanimal, the body weight of the animal, as well as the severity and stageof the cancer.

A suitable dose is that which will result in a concentration of theactive agent in tumor tissue which is known to effect the desiredresponse. The preferred dosage is the amount which results in maximuminhibition of cancer cell growth, without unmanageable side effects.Administration of API-2 (or a pharmaceutically acceptable salt thereof)can be continuous or at distinct intervals, as can be determined by aperson of ordinary skill in the art.

Mammalian species which benefit from the disclosed methods for theinhibition of cancer cell growth, include, but are not limited to,primates, such as apes, chimpanzees, orangutans, humans, monkeys;domesticated animals (e.g., pets) such as dogs, cats, guinea pigs,hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales. Theterms “patient” and “subject” are used herein interchangeably and areintended to include such human and non-human mammalian species.Likewise, in vitro methods of the present invention can be earned out oncells of such mammalian species.

Patients in need of treatment using the methods of the present inventioncan be identified using standard techniques known to those in themedical profession.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 In Vitro Screening Materials

Cell Lines and NCI Diversity Set. All cell lines used in this study wereeither purchased from ATCC or described previously (Cheng, J. Q., et al.Oncogene, 14: 2793-2801, 1997, West, K. A., et al. Drug Resist. Updat.,5: 234-248, 2002, Satyamoorthy, K., et al. Cancer Res. 61: 7318-7324,2001). The NCI Structural Diversity Set is a library of 1,992 compoundsselected from the approximately 140,000-compound NCI drug depository.In-depth data on the selection, structures, and activities of thesediversity set compounds can be found on the NCI DevelopmentalTherapeutics Program web site.

Screening for Inhibition of Akt-transformed Cell Growth. AKT2transformed NIH3T3 cells or LXSN vector-transfected NIH3T3 control cells(Cheng, J. Q., et al. Oncogene, 14: 2793-2801, 1997) were plated into96-well tissue culture plate. Following treatment with 5 μM of NCIDiversity Set compound, cell growth was detected with CellTier 96 OneSolution Cell Proliferation kit (Promega). Compounds that inhibit growthin AKT2-transformed but not LXSN-transfected NIH3T3 cells wereconsidered as candidates of Akt inhibitor and subjected to furtheranalysis.

In vitro Protein Kinase, Cell Survival and Apoptosis Assays. In vitrokinase was performed as previously described (see, for example, Jiang,K., Coppola, et al. Mol. Cell. Biol., 20:139-148, 2000). Cell survivalwas assayed with MTS (Promega). Apoptosis was detected with annexin V,which was performed as previously described (Jiang, K., Coppola, et al.Mol. Cell. Biol., 20:139-148, 2000). Recombinant Akt and PDK1 werepurchased from Upstate Biotechnology Inc.

Results

Identification of Small Molecule Inhibitor of Akt Signaling Pathway,API-2. Frequent alterations of Akt have been detected in human cancerand disruption of Akt pathway induces apoptosis and inhibits tumorgrowth (Jetzt, A., et al. Cancer Res., 63: 697-706, 2003). Thus, Akt hasbeen considered as an attractive molecular target for development ofnovel cancer therapeutics. To identify small molecule inhibitor(s) ofAkt, a chemical library of 1,992-compounds from the NCI (the NCIDiversity Set) was evaluated for agents capable of inhibition of growthin AKT2-transformed but not empty vector LXSN-transfected NIH3T3 cellsas described in “Materials and Methods”. Repeated experiments showedthat 32 compounds inhibited growth only in AKT2-transformaed cells. Themost potent of these compounds, API-2 (NCI identifier: NSC 154020),suppressed cell growth at a concentration of 50 nM. FIG. 1A shows thechemical structure of API-2, which is also known as triciribine(Schweinsberg, P. D., et al. Biochem Pharmacol., 30: 2521-2526, 1981).The fact that API-2 inhibited selectively AKT-2 transformed cells overuntransformed parental cells prompted us to determine whether API-2 isan inhibitor of AKT2 kinase. To this end, AKT2 was immunoprecipitatedwith anti-AKT2 antibody from AKT-2 transformed NIH3T3 cells followingtreatment with API-2. AKT2 immunoprecipitates were immunoblotted withanti-phospho-Akt antibodies. As shown in FIG. 1B, API-2 significantlyinhibited AKT2 phosphorylation at both threonine-309 and serine-474,which are required for full activation of AKT2 (Datta, S. R., et al.Genes Dev. 13: 2905-2927, 1999). As three isoforms of Akt share highhomology and similar structure, the effect of API-2 on their kinaseactivities was evaluated. HEK293 cells were transfected with HA-Akt1,-AKT2 and -AKT3, serum-starved overnight and treated with API-2 for 60min prior to EGF (50 ng/ml) stimulation. Triple experiments showed thatAPI-2 suppressed EGF-induced kinase activity and phosphorylation ofAkt1, AKT2 and AKT3 (FIG. 1C). However, kinase activity of recombinantconstitutively active AKT2 (Myr-AKT2) was not inhibited by API-2 in anin vitro kinase reaction (FIG. 1D), suggesting that API-2 does notdirectly inhibit Akt in vitro and that API-2 neither functions as ATPcompetitor nor as the substrate competitor that binds to active site ofAkt.

API-2 Does Not Inhibit Known Upstream Activators of Akt. It has beenwell documented that Akt is activated by extracellular stimuli andintracellular signal molecules, such as active Ras and Src, through aPI3K-dependent manner. Therefore, API-2 inhibition of Akt could resultfrom targeting upstream molecule(s) of Akt. As PI3K and PDK1 are directupstream regulators of Akt (Datta, S. R., et al. Genes Dev. 13:2905-2927, 1999), whether API-2 inhibits PI3K and/or PDK1 was examined.HEK293 cells were serum-starved and then treated with API-2 or PI3Kinhibitor, wortmannin, for 30 min prior to EGF stimulation. PI3K wasimmunoprecipitated with anti-p100α antibody. The immunoprecipitates weresubjected to in vitro PI3K kinase assay using PI-4-P as a substrate. Asshown in FIG. 2A, the EGF-induced PI3K activity was inhibited bywortmannin but not by API-2. To evaluate the effect of API-2 on PDK1, anassay in which recombinant PDK1 promotes the threonine-309phosphorylation of AKT2 peptides was used in the presence of lipidvesicles containing phosphotidylinositol. As shown in FIG. 2B, the assaywas potently inhibited by the control PDK1 inhibitor staurosporine(IC50=5 nM). In contrast, API-2 displayed only 21% inhibition of theassay at the highest concentration tested (5.1 μM). These datademonstrate that API-2 is not a potent inhibitor of PDK1. To furtherevaluate the effect of API-2 on PDK1 activation, the autophosphorylationlevel of PDK1 at serine-241, a residue that is phosphorylated by itselfand is critical for its activity was examined (Datta, S. R., et al.Genes Dev. 13: 2905-2927, 1999), following API-2 treatment of HEK293cells. Triplicate experiments show that phosphorylation levels of PDK1were not inhibited by API-2 (FIG. 2B). However, PI3K inhibitorwortmannin, as expected, inhibited EGF-stimulated PDK1 (FIG. 2B).

API-2 Is Highly Selective for the Akt over PKC, PKA, SGK, STAT, JNK,p38, and ERK Signaling Pathways. Akt belongs to AGC (PKA/PKG/PKC) kinasefamily, which also include PKA, PKC, serum- and glucocorticoid-induciblekinase (SGK), p90 ribosomal S6 kinase, p70^(S6K), mitogen- andstress-activated protein kinase and PKC-related kinase. Among AGC kinasefamily, protein structures of PKA, PKC and SGK are more close to Aktkinase than other members. Therefore, next examined were the effects ofAPI-2 on the enzymatic activities of these 3 kinases. HEK293 cells weretransfected with HA-tagged PKA, PKC□□or SGK. In vitro kinase assay andimmunoblotting analysis showed that the kinase activities of PKA andPKCα were inhibited by PKAI and Ro 31-8220, a PKC inhibitor,respectively, whereas API-2 exhibited no effect on their activities(FIGS. 2C and 2E). Further, serum-induced SGK kinase activity wasattenuated by wortmannin but not by API-2 (FIG. 2D). In addition, it wasdetermined whether API-2 has effect on other oncogenic survivalpathways. Western blotting analyses with commercially availableanti-phospho-antibodies revealed that phosphorylation levels of Stat3,JNK, p38 and Erk½ were not affected by API-2 treatment (FIG. 2F). Thesedata indicate that API-2 specifically inhibits Akt signaling pathway.

API-2 Suppresses Cell Growth and Induces Apoptosis inAkt-overexpressing/activating Human Cancer Cell Lines. The ability ofAPI-2 to selectively inhibit the Akt pathway suggests that it shouldinhibit proliferation and/or induces apoptosis preferentially in thosetumor cells with aberrant expression/activation of Akt. As activation ofAkt in human malignancies commonly results from overexpression of Akt orPTEN mutations, API-2 was used to treat the cells that expressconstitutively active Akt, caused by overexpression of AKT2 (OVCAR3,OVCAR8, PANC1 and AKT2-transformed NIH3T3) or mutations of the PTEN gene(PC-3, LNCaP, MDA-MB-468), and cells that do not (OVCAR5, DU-145, T47D,COLO357 and LXSN-NIH3T3) as well as melanoma cells that are activated byIGF-1 to activate Akt or do not respond to growth stimulation by IGF-1(Satyamoorthy, K., et al. Cancer Res. 61: 7318-7324, 2001).Immunoblotting analysis showed that phosphorylation levels of Akt wereinhibited by API-2 only in the cells expressing elevated Akt orresponding to IGF-1 simulation (FIG. 3A). Accordingly, API-2 inhibitedcell growth to a much higher degree in Akt-overexpressing/activatingcells as compared to those with low levels of Akt. As shown in FIG. 3B,API-2 treatment inhibited cell proliferation by approximate 50-60% inAkt-overexpressing/activating cell lines, LNCaP, PC-3, OVCAR3, OVCA8,PANC1, MDA-MB-468, and WM35, whereas only by about 10-20% in DU145,OVCAR5, COLO357, T47D and WM852 cells, which exhibit low levels of Aktor do not respond to growth stimulation by IGF-1. Moreover, API-2induces apoptosis by 8-fold (OVCAR3), 6-fold (OVCAR8), 6-fold (PANC1),and 3-fold (AKT2-NIH3T3). No significant difference of apoptosis wasobserved between API-2 and vehicle (DMSO) treatment in OVCAR5, COLO357and LXSN-NIH3T3 cells. Thus, API-2 inhibits cell growth and inducesapoptosis preferentially in cells that express aberrant Akt.

API-2 Inhibits Downstream Targets of Akt. It has been shown that Aktexerts its cellular effects through phosphorylation of a number ofproteins (Datta, S. R., et al. Genes Dev. 13: 2905-2927, 1999). Morethan 20 proteins have been identified as Akt substrates, including themembers of Forkhead protein family (FKHR, AFX and FKHRL1),tuberlin/TSC2, p70^(S6K), GSK-3β, p21^(WAF1/Cip1), p27^(kip1), MDM2,Bad, ASK1 and IKKα□etc. It was next examined whether API-2 inhibitsdownstream targets of Akt. As anti-phospho-tuberlin, -Bad, -AFX, and-GSK-3β antibodies are commercially available, therefore, the effect ofAPI-2 on their phosphorylation induced by Akt was determined. FollowingAPI-2 (1 □M) treatment, OVCAR3 cells were lysed and immunoblotted withthe individual anti-phospho-antibody. FIG. 4A shows that API-2considerably inhibited the phosphorylation levels of tuberlin leading tostabilization and upregulation of tuberin (Dan, H. C., et al. J. Biol.Chem., 277: 35364-35370, 2002). The phosphorylation levels of Bad,GSK-3β, and AFX were partially attenuated by API-2. These data suggestthat API-2 induces cell death and cell growth arrest by inhibitingphosphorylation of its downstream targets. API-2 inhibition of Aktdownstream targets at different degrees could be due to the fact thatphosphorylation sites of these targets are also regulated by otherkinase(s), for instance, Bad serine-136 is phosphorylated by PAK1 inaddition to Akt (Schurmann, A., et al. Mol. Cell. Biol., 20: 453-461,2000).

Example 2 Antitumor Activity in the Nude Mouse Tumor Xenograft Model

Tumor cells were harvested, resuspended in PBS, and injected s.c. intothe right and left flanks (2×10⁶ cells/flank) of 8-week-old female nudemice as reported previously (Sun, J., Blaskovic, et al. Cancer Res., 59:4919-4926, 1999). When tumors reached about 100-150 mm³, animals wererandomized and dosed i.p. with 0.2 ml vehicle of drug daily. Controlanimals were received DMSO (20%) vehicle, whereas treated animals wereinjected with API-2 (1 mg/kg/day) in 20% DMSO.

API-2 Inhibits the Growth of Tumors in Nude Mice that Overexpress Akt.Frequent overexpression/activation and/or amplification of AKT1 and AKT2in human ovarian and pancreatic cancer was shown (Cheng, J. Q., andNicosia, S. V. AKT signal transduction pathway in oncogenesis. In SchwabD, Editor, Encyclopedic Reference of Cancer. Berlin Heidelberg and NewYork: Springer; 2001. pp 35-7). Inhibition of Akt pathway by inhibitorsof PI3K, HSP70, Src and farnesyltransferase resulted in cell growtharrest and induction of apoptosis (Solit, D. B., et al. Cancer Res., 63:2139-2144, 2003, Xu, W., et al. Cancer Res., 63: 7777-7784, 2003). Arecent study showed that the tumor growth of xenografts with elevatedAkt was also significantly inhibited by intratumoral injection ofadenovirus of dominant negative Akt (Jetzt, A., et al. Cancer Res., 63:697-706, 2003). Because API-2 inhibits Akt signaling and inducesapoptosis and cell growth arrest only in cancer cells with elevatedlevels of Akt (FIG. 3), the growth of tumors with elevated levels of Aktshould be more sensitive to API-2 than that of tumors with low levels ofAkt in nude mice. To this end, s.c. Akt-overexpressing cells (OVCAR3,OVCAR8 and PANC-1) were s.c. implanted into the right flank, and thosecell lines that express low levels of Akt (OVCAR5 and COLO357) into theleft flank of mice. When the tumors reached an average size of about100-150 mm³, the animals were randomized and treated i.p. with eithervehicle or API-2 (1 mg/kg/day). As illustrated in FIG. 4B, OVCAR-5 andCOLO357 tumors treated with vehicle grew to about 800-1,000 mm³ 49 daysafter tumor implantation. OVCAR3, OVCAR8 and PANC1 tumors treated withvehicle control grew to about 700-900 mm³ 49 days after tumorimplantation. API-2 inhibited OVCAR3, OVCAR8 and PANC1 tumor growth by90%, 88% and 80%, respectively. In contrast, API-2 had little effect onthe growth of OVCAR5 and COLO357 cells in nude mice (FIGS. 4B-4D anddata not shown). At dose 1 mg/kg/day, API-2 had no effects on bloodglucose level, body weight, activity and food intake of mice. In treatedtumor samples, Akt activity was inhibited by API-2 without change oftotal Akt content (FIG. 4E). Taken together, these results indicate thatAPI-2 selectively inhibits the growth of tumors with elevated levels ofAkt.

Example 3 TCN Directly Inhibits Wild Type Akt Kinase Activity

API-2 (TCN) can directly inhibit wild type Akt kinase activity inducedby PDK1 in vitro (FIG. 1). This result supports that API-2 is a directAkt inhibitor and that the underlying mechanism may be API-2 binding toPH domain and/or threonine-308 of Akt. An in vitro kinase assay wasperformed with recombinant of PDK1 and Akt in a kinase buffer containingphosphatidylinositol-3,4,5-P3 (PIP3), API-2 and histone H2B assubstrate. After incubation of 30 min, the reactions were separated bySDS-PAGE and exposed in a film.

Example 4 TCN is Effective in Cancer Resistant Cells

The effects of TCN (API-2) were tested in cisplatin, paclitaxel, andtamoxifen resistant A270CP, C-13, OVCAR433 and MCF7/TAM cells. API-2overcame cisplatin, paclitaxel, and tamoxifen resistance in these cells

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention will beevident to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of thisinvention.

1-42. (canceled)
 43. A method for treating esophageal adenocarcinoma ina mammal comprising administering an effective amount of a compound offormula:

wherein each R₂′ and R₃′ are independently hydrogen, optionallysubstituted phosphate or phosphonate; acyl; alkyl; amide, sulfonateester; sulfonyl, methanesulfonyl and benzylsulfonyl, wherein the phenylgroup is optionally substituted with one or more substituents;optionally substituted arylsulfonyl; a lipid, phospholipid; an aminoacid; a carbohydrate; a peptide; or cholesterol; or otherpharmaceutically acceptable leaving group that, in vivo, provides acompound wherein R₂′ and R₃′ is independently H or mono-, di- ortri-phosphate; wherein R^(x) and R^(y) are independently hydrogen,optionally substituted phosphate; acyl; amide, alkyl; aromatic,polyoxyalkylene, polyethyleneglycol, arylsulfonyl; a lipid, aphospholipid; an amino acid; a carbohydrate; a peptide; or cholesterol;or other pharmaceutically acceptable leaving group; R₁ and R₂ each areindependently H, optionally substituted straight chained, branched orcyclic alkyl, alkenyl, or alkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl,CO-aryl or heteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substitutedaryl, sulfonyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.
 44. Themethod of claim 43, wherein the subject has been diagnosed withesophogeal adenocarcinoma.
 45. The method of claim 43, wherein thecompound is administered one time per week for three weeks followed by aone week period wherein the compound is not administered.
 46. The methodof claim 45, wherein the administration is repeated at least twice. 47.The method of claim 43, wherein at least 10 mg/m² of the compound isadministered.
 48. The method of claim 43, wherein 10 mg/m² or less ofthe compound is administered.
 49. The method of claim 43, wherein thethe drug is administered intravenously.
 50. The method of claim 43,wherein the mammal is a human.
 51. A method to treat esophogealadenocarcinoma in a mammal comprising administering to said mammal adose of 10 mg/m² or less of a compound of the formula:

wherein each R₂′ and R₃′ are independently hydrogen, optionallysubstituted phosphate or phosphonate; acyl; alkyl; amide, sulfonateester; sulfonyl, methanesulfonyl and benzylsulfonyl, wherein the phenylgroup is optionally substituted with one or more substituents;optionally substituted arylsulfonyl; a lipid, a phospholipid; an aminoacid; a carbohydrate; a peptide; or cholesterol; or otherpharmaceutically acceptable leaving group that, in vivo, provides acompound wherein R₂′ and R₃″ is independently H or mono-, di- ortri-phosphate; wherein R^(x) and R^(y) are independently hydrogen,optionally substituted phosphate; acyl; amide, alkyl; aromatic,polyoxyalkylene, polyethyleneglycol, arylsulfonyl; a lipid, aphospholipid; an amino acid; a carbohydrate; a peptide; or cholesterol;or other pharmaceutically acceptable leaving group; and wherein R₁ andR₂ each are independently H, optionally substituted straight chained,branched or cyclic alkyl, alkenyl, or alkynyl, CO-alkyl, CO-alkenyl,CO-alkynyl, CO-aryl or heteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl,CO-substituted aryl, sulfonyl, alkylsulfonyl, arylsulfonyl,aralkylsulfonyl.
 52. The method of claim 51, wherein the administrationis repeated weekly for four weeks, and wherein the four week period isfollowed by a one week period wherein no drug is administered.
 53. Themethod of claim 51, wherein the administration represents a dosingcycle.
 54. The method of claim 53, wherein the dosing cycle is repeatedat least twice.
 55. The method of claim 53, wherein the dosing cycle isrepeated until regression of the cancer is achieved.
 56. The method ofclaim 51, wherein the drug is administered intravenously.
 57. The methodof claim 51, wherein the mammal is a human.